U.S. patent number 7,097,834 [Application Number 09/211,315] was granted by the patent office on 2006-08-29 for osteoprotegerin binding proteins.
This patent grant is currently assigned to Amgen Inc.. Invention is credited to William J. Boyle.
United States Patent |
7,097,834 |
Boyle |
August 29, 2006 |
Osteoprotegerin binding proteins
Abstract
A novel polypeptide, osteoprotegerin binding protein, involved
in osteolcast maturation has been identified based upon its
affinity for osteoprotegerin. Nucleic acid sequences encoding the
polypeptide, or a fragment, analog or derivative thereof, vectors
and host cells for production, methods of preparing osteoprotegerin
binding protein, and binding assays are also described.
Compositions and methods for the treatment of bone diseases such as
osteoporosis, bone loss due to arthritis or metastasis,
hypercalcemia, and Paget's disease are also provided.
Inventors: |
Boyle; William J. (Moorpark,
CA) |
Assignee: |
Amgen Inc. (Thousand Oaks,
CA)
|
Family
ID: |
27126366 |
Appl.
No.: |
09/211,315 |
Filed: |
December 14, 1998 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
08880855 |
Jun 23, 1997 |
|
|
|
|
08842842 |
Apr 16, 1997 |
5843678 |
|
|
|
Current U.S.
Class: |
424/130.1;
424/136.1; 424/134.1; 424/142.1; 424/141.1; 424/139.1;
424/133.1 |
Current CPC
Class: |
A61P
19/08 (20180101); A61P 1/02 (20180101); A61P
19/02 (20180101); A61P 29/00 (20180101); C07K
14/70575 (20130101); C07K 14/70578 (20130101); A61P
35/04 (20180101); A61P 19/00 (20180101); A61P
3/14 (20180101); A61P 19/10 (20180101); C07K
2319/30 (20130101); A61K 38/00 (20130101); A61K
39/00 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); A61K 39/44 (20060101) |
Field of
Search: |
;536/24.5 ;514/44
;530/387.3,387.7,388.1,388.15,388.2
;424/133.1,134.1,138.1,139.1,141.1,142.1,143.1,130.1,136.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0816380 |
|
Jan 1998 |
|
EP |
|
0873998 |
|
Oct 1998 |
|
EP |
|
0 911 342 |
|
Apr 1999 |
|
EP |
|
11009269 |
|
Jan 1999 |
|
JP |
|
WO 86/00922 |
|
Feb 1986 |
|
WO |
|
WO 90/14363 |
|
Nov 1990 |
|
WO |
|
WO93/12227 |
|
Jun 1993 |
|
WO |
|
WO96/26271 |
|
Aug 1996 |
|
WO |
|
WO 96/34095 |
|
Oct 1996 |
|
WO |
|
WO 97/23614 |
|
Jul 1997 |
|
WO |
|
WO 98/25958 |
|
Jun 1998 |
|
WO |
|
WO 98 25958 |
|
Jun 1998 |
|
WO |
|
WO 98/28423 |
|
Jul 1998 |
|
WO |
|
WO 98 28426 |
|
Jul 1998 |
|
WO |
|
WO 98/54201 |
|
Dec 1998 |
|
WO |
|
WO 99/29865 |
|
Jun 1999 |
|
WO |
|
WO 99/65449 |
|
Dec 1999 |
|
WO |
|
WO 99/65495 |
|
Dec 1999 |
|
WO |
|
WO 01/23549 |
|
Apr 2001 |
|
WO |
|
WO 02/15846 |
|
Feb 2002 |
|
WO |
|
Other References
Suda T et al, Modulation of Osteoclast Differentiatio by Local
Factors, Bone, vol. 17, 87S-91S, Aug. 1, 1995. cited by examiner
.
Cooke et al, An overview of progress in antisense therapeutics,
Antisense & Nucleic Acid Drug Development, 8:115-122, Jan. 1,
1998. cited by examiner .
Takahashi, Biochem. Biophys. Res. Comm. 256, 449-455, 1999. cited
by examiner .
Goh et al., Protein Engineering 4, 785-791 (1991). cited by other
.
Banner et al., Cell 73, 431-445 (1993). cited by other .
Chomczynski and Sacchi. Anal. Biochem. 162, 156-159, (1987). cited
by other .
Goeddel, D.V. ed., Methods in Enzymology v. 185, Academic Press
(1990). cited by other .
Gribskov et al. Proc. Natl. Acad. Sci. USA 83, 4355-4359 (1987).
cited by other .
Jimi et al., Endocrinology 137, 2187-2190 (1996). cited by other
.
Luethy et al. Protein Sci. 3, 139-146 (1994). cited by other .
Nagata and Golstein, Science 267, 1449-1456 (1995). cited by other
.
Pearson, Meth. Enzymol. 183, 63-98 (1990). cited by other .
Remington's Pharmaceutical Sciences, 18th ed. A.R. Gennaro, ed.
Mack, Easton, PA (1980). cited by other .
Sambrook et al. Molecular Cloning: A Laboratory Manual Cold Spring
Harbor Press, New York (1989). cited by other .
Jones et al., J. Cell. Sci. Suppl. 13, 11-18 (1990). cited by other
.
Smith et al., Cell 76, 959-962 (1994). cited by other .
Wiley et al. Immunity 3, 673-382 (1995). cited by other .
Yasuda et al., Proceeds of the National Academy of Sciences of USA
95, 3597-3602 (1998). cited by other .
E.M.B.L. Databases Accession No. AA170348 (1997). cited by other
.
* Lacey et al., Cell 93: 165-176 (1998). cited by other .
* Wong, et al, J. Biol. Chem. 272: 25190-25194 (1997). cited by
other .
* Anderson et al., Nature 390: 175-179 (1997). cited by other .
Tsukii et al, "Osteoclast Differentiation Factor Mediates an
Essential Signal for Bone Resorption . . . ", Biochemical and
Biophysical Research Communications, 246, pp 337-341 (1998). cited
by other .
U.S. Appl. No. 09/957,944, filed Sep. 20, 2001, Dougall. cited by
other .
U.S. Appl. No. 10/151,071, filed May 17, 2002, Dougall et al. cited
by other .
U.S. Appl. No. 10/166,232, filed Jun. 5, 2002, Dougall. cited by
other .
U.S. Appl. No. 10/405,878, filed Apr. 1, 2003, Anderson. cited by
other .
Boyle v. Gorman and Mattson, Board of Patent Appeals and
Interferences, Interference No. 104,336, Paper No. 39. cited by
other .
Camerini et al., J. Immunol., "The T Cell Activation Antigen CD27
is a Member of the Nerve Growth Factor/Tumor Necrosis Factor
Receptor Gene Family", 147:3165-3169 (1991). cited by other .
Caux et al., J. Exp. Med., "Activation of Human Dendritic Cells
through CD40 Cross-linking", 180:1263-1272 (1994). cited by other
.
Durkop et al., Cell, "Molecular Cloning and Expression of a New
Member of the Nerve Growth Factor Receptor Family That Is
Characteristic for Hodgkin's Disease", 68:421-427 (1992). cited by
other .
EMBL-EBI Database Entry HS421358, Accession No. W74421, Homo
sapiens cDNA Clone IMAGE:346544 3' Similar to Contains Alu
Repetitive Element, Hillier et al., (Jun. 1996). cited by other
.
Galibert et al., J. Biol. Chem., "The Involvement of Multiple Tumor
Necrosis Factor Receptor (TNFR)-Associated Factors in the Signaling
Mechanisms of Receptor Activator of NF-.kappa.B, a Member of the
TNFR Superfamily", 273(51):34120-34127 (1998). cited by other .
Gray et al., Genetics, "P-Element-Induced Recombination in
Drosophila melanogaster. Hybrid Element Insertion",
144(4):1601-1610 (1996). cited by other .
Itoh et al., Cell, "The Polypeptide Encoded by the cDNA for Human
Cell Surface Antigen Fas Can Mediate Apoptosis", 66:233-243 (1991).
cited by other .
Johnson et al., Cell, "Expression and Structure of the Human NGF
Receptor", 47:545-554 (1986). cited by other .
Kodaira et al., Gene, "Cloning and characterization of the gene
encoding mouse osteoclast differentiation factor", 230:121-127
(1999). cited by other .
Kwon et al., Proc. Natl. Acad. Sci. USA, "cDNA sequences of two
inducible T-cell genes", 86:1963-1967 (1989). cited by other .
Mallett et al., EMBO J., "Characterization of the MRC OX40 antigen
of activated CD4 positive T lymphocytes--a molecule related to
nerve growth factor receptor", 9:1063-1068 (1990). cited by other
.
NCBI, Marra et al., The WashU-HHMI Mouse EST Project, GenBank
Accession No. AA170348, (Feb. 16, 1997). cited by other .
Nakagawa et al., Biochem. Biophys. Res. Commun., "RANK is the
Essential Signaling Receptor for Osteoclast Differentiation Factor
in Osteoclastogenesis", 253:395-400 (1998). cited by other .
Romani et al., J. Exp. Med., "Proliferating Dendritic Cell
Progenitors in Human Blood", 180:83-93 (1994). cited by other .
Rothe, M. et al., Cell, "The TNFR2-TRAF Signaling Complex Contains
Two Novel Proteins Related to Baculoviral Inhibitor of Apoptosis
Proteins", 83:1243-1252 (1995). cited by other .
Schall, et al., Cell, "Molecular Cloning and Expression of a
Receptor for Human Tumor Necrosis Factor", 61:361-370 (1990). cited
by other .
Simonet et al., Cell, "Osteoprotegerin: A Novel Secreted Protein
Involved in the Regulation of Bone Density", 89:309-319 (1997).
cited by other .
Stamenkovic et al., EMBO J., "A B-lymphocyte activation molecule
related to the nerve growth factor receptor and induced by
cytokines in carcinomas", 8:1403-1410 (1989). cited by other .
Suda et al., Endocr. Rev., "Modulation of Osteoclast
Differentiation", 13:66-80 (1992). cited by other .
Suda et al., Bone, "Modulation of Osteoclast Differentiation by
Local Factors", 17(2):87S-91S (1995). cited by other .
Suda et al., Endocr. Rev., Monograph, "Modulation of Osteoclast
Differentiation: Update 1995", 4(1):266-270 (1995). cited by other
.
Viney et al., J. Immunol., "Expanding Dendritic Cells In Vivo
Enhances the Induction of Oral Tolerance", 160:5815-5825 (1998).
cited by other .
Wong et al., J. Biol. Chem. "The TRAF Family of Signal Transducers
Mediates NF-.kappa.B Activation by the TRANCE Receptor",
273(43):28355-28359 (1998). cited by other .
Wong et al., J. Exp. Med., "TRANCE (Tumor Necrosis Factor
[TNF]-related Activation-induced Cytokine), a New TNF Family Member
Predominantly Expressed in T cells, Is a Dendritic Cell-specific
Survival Factor", 186:2075-2080 (1997). cited by other .
Xu et al., Immunity, "Targeted Disruption of TRAF3 Leads to
Postnatal Lethality and Defective T-Dependent Immune Responses",
5:407-415 (1996). cited by other .
Yun et al., Immunol., "OPG/FDCR-1, a TNF Receptor Family Member, Is
Expressed in Lymphoid Cells and Is Up-Regulated by Ligating
CD40.sup.1", 161:6113-6121 (1998). cited by other .
Jakovits, Aya, "Production of fully human antibodies by transgenic
mice", Current Opinion in Biotechnology, 6:561-566, 1995. cited by
other .
Lonberg, Nils et al., "Human Antibodies from Transgenic Mice",
Intern. Rev. Immunol., vol. 13, pp. 65-93, 1995. cited by
other.
|
Primary Examiner: Kemmerer; Elizabeth
Attorney, Agent or Firm: Winter; Robert B.
Parent Case Text
This application is a continuation of application Ser. No.
08/880,855, filed Jun. 23, 1997, now abandoned, which is a
continuation-in-part of application Ser. No. 08/842,842, filed Apr.
16, 1997, now U.S. Pat. No. 5,843,678, which is hereby incorporated
by reference.
Claims
What is claimed is:
1. A method of inhibiting bone resorption in a mammal in need
thereof comprising administering to the mammal an antagonist
antibody or binding fragment thereof which binds to the
osteoprotegerin binding protein of SEQ ID NO:39.
2. The method of claim 1 wherein the antibody is a monoclonal
antibody or binding fragment thereof.
3. The method of claim 1 wherein the antibody is a recombinant
antibody or binding fragment thereof.
4. The method of claim 3 wherein the antibody or fragment is a
chimeric antibody or a CDR-grafted antibody or a binding fragment
thereof.
5. The method of claim 1 wherein the antibody is a human antibody
or binding fragment thereof.
6. The method of claim 5 wherein the antibody is prepared by
immunization of a transgenic animal capable of producing human
antibodies.
7. The method of claim 1 wherein the antibody or binding fragment
thereof binds to an epitope on the extracellular domain or to an
epitope on a fragment of the extracellular domain of an
osteoprotegerin binding protein.
8. The method of claim 7 wherein the epitope comprises the BB' loop
of an osteoprotegerin binding protein.
9. The method of claim 7 wherein the epitope comprises the EF loop
of an osteoprotegerin binding protein.
10. The method of claim 1 wherein the antibody or binding fragment
further comprises a composition comprising a pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative
and/or adjuvant.
11. The method of any of claims 1, 2, 3, 4, 5, 7, or 10 further
comprising administering one or more of a bone morphogenic factor,
transforming growth factor-.beta., a transforming growth
factor-.beta. family member, a fibroblast growth factor, an
interleukin-1 inhibitor, a TNF.alpha. inhibitor, a parathyroid
hormone, an E series prostaglandin, a bisphosphonate, or a
bone-enhancing mineral.
12. The method of any of claims 1, 2, 3, 4, 5, 7, or 10 or wherein
bone resorption is associated with a bone disease selected from
osteoporosis, osteomyelitis, hypercalcemia, osteopenia brought on
by surgery or steroid administration, Paget's disease,
osteonecrosis, bone loss due to rheumatoid arthritis, periodontal
bone loss, osteopenia due to immobilization, prosthetic loosening
and osteolytic metastasis.
13. The method of claim 1 wherein the antibody or binding fragment
thereof binds to a membrane associated form of osteoprotegerin
binding protein.
14. The method of claim 1 wherein the antibody or binding fragment
thereof binds to a soluble osteoprotegerin binding protein.
15. A method of inhibiting osteoclastogenesis in a mammal in need
thereof comprising administering to the mammal an antagonist
antibody or binding fragment thereof which binds to the
osteoprotegerin binding protein of SEQ ID NO:39.
16. The method of claim 15 wherein the antibody is a monoclonal
antibody or binding fragment thereof.
17. The method of claim 15 wherein the antibody is a recombinant
antibody or binding fragment thereof.
18. The method of claim 15 wherein the antibody is a chimeric
antibody or a CDR-grafted antibody.
19. The method of claim 15 wherein the antibody is a human antibody
or binding fragment thereof.
20. The method of claim 19 wherein the antibody is prepared by
immunization of a transgenic animal capable of producing human
antibodies.
21. The method of claim 15 wherein the antibody or binding fragment
thereof binds to an epitope on the extracellular domain or to an
epitope on a fragment of the extracellular domain of an
osteoprotegerin binding protein.
22. The method of claim 21 wherein the epitope comprises the BB'
loop of an osteoprotegerin binding protein.
23. The method of claim 21 wherein the epitope comprises the EF
loop of an osteoprotegerin binding protein.
24. The method of claim 15 wherein the antibody or binding fragment
thereof binds to a membrane associated form of osteoprotegerin
binding protein.
25. The method of claim 15 wherein the antibody or binding fragment
thereof binds to a soluble osteoprotegerin binding protein.
26. The method of claim 15 wherein the antibody or binding fragment
further comprises a composition comprising a pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative
and/or adjuvant.
27. The method of any of claims 15, 16, 17, 18, 19, 21, 24, 25, or
26 further comprising administering one or more of a bone
morphogenic factor, transforming growth factor-.beta., a
transforming growth factor-.beta. family member, a fibroblast
growth factor, an interleukin-1 inhibitor, a TNF.alpha. inhibitor,
a parathyroid hormone, an E series prostaglandin, a bisphosphonate,
or a bone-enhancing mineral.
28. The method of any of claims 15, 16, 17, 18, 19, 21, 24, 25, or
26 wherein osteoclastogenesis is associated with a condition
selected from osteoporosis, osteomyelitis, hypercalcemia,
osteopenia brought on by surgery or steroid administration, Paget's
disease, osteonecrosis, bone loss due to rheumatoid arthritis,
periodontal bone loss, osteopenia due to immobilization, prosthetic
loosening and osteolytic metastasis.
29. The method of claims 1 or 15 wherein the mammal is a human.
30. The method of claims 1 or 15 wherein the antibody is raised
against an osteoprotegerin binding protein comprising the amino
acid sequence of SEQ ID NO:39 or an antigenic fragment thereof.
31. The method of claim 1 or 15 wherein the antibody is raised
against an osteoprotegerin binding protein comprising the amino
acid sequence of SEQ ID NO:39 from residues 69 317.
Description
FIELD OF THE INVENTION
The present invention relates to polypeptides which are involved in
osteoclast differentiation. More particularly, the invention
relates to osteoprotegerin binding proteins, nucleic acids encoding
the proteins, expression vectors and host cells for production of
the proteins, and binding assays. Compositions and methods for the
treatment of bone diseases, such as osteoporosis, bone loss from
arthritis, Paget's disease, and hypercalcemia, are also
described.
BACKGROUND OF THE INVENTION
Living bone tissue exhibits a dynamic equilibrium between
deposition and resorption of bone. These processes are mediated
primarily by two cell types: osteoblasts, which secrete molecules
that comprise the organic matrix of bone; and osteoclasts, which
promote dissolution of the bone matrix and solubilization of bone
salts. In young individuals with growing bone, the rate of bone
deposition exceeds the rate of bone resorption, while in older
individuals the rate of resorption can exceed deposition. In the
latter situation, the increased breakdown of bone leads to reduced
bone mass and strength, increased risk of fractures, and slow or
incomplete repair of broken bones.
Osteoclasts are large phagocytic multinucleated cells which are
formed from hematopoietic precursor cells in the bone marrow.
Although the growth and formation of mature functional osteoclasts
is not well understood, it is thought that osteoclasts mature along
the monocyte/macrophage cell lineage in response to exposure to
various growth-promoting factors. Early development of bone marrow
precursor cells to preosteoclasts are believed to mediated by
soluble factors such as tumor necrosis factor-.alpha.
(TNF-.alpha.), tumor necrosis factor-.beta. (TNF-.beta.),
interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6),
and leukemia inhibitory factor (LIF). In culture, preosteoclasts
are formed in the presence of added macrophage colony stimulating
factor (M-CSF). These factors act primarily in early steps of
osteoclast development. The involvement of polypeptide factors in
terminal stages of osteoclast formation has not been extensively
reported. It has been reported, however, that parathyroid hormone
stimulates the formation and activity of osteoclasts and that
calcitonin has the opposite effect, although to a lesser
extent.
Recently, a new polypeptide factor, termed osteoprotegerin (OPG),
has been described which negatively regulated formation of
osteoclasts in vitro and in vivo (see co-owned and co-pending U.S.
Ser. No. 08/577,788 filed Dec. 22, 1995, Ser. No. 08/706,945 filed
Sep. 3, 1996, and Ser. No. 08/771,777, filed Dec. 20, 1996, now
abandoned, hereby incorporated by reference; and PCT Application
No. WO96/26271). OPG dramatically increased the bone density in
transgenic mice expressing the OPG polypeptide and reduced the
extent of bone loss when administered to ovariectomized rats. An
analysis of OPG activity in in vitro osteoclast formation revealed
that OPG does not interfere with the growth and differentiation of
monocyte/macrophage precursors, but more likely blocks the
differentiation of osteoclasts from monocyte/macrophage precursors.
Thus OPG appears to have specificity in regulating the extent of
osteoclast formation.
OPG comprises two polypeptide domains having different structural
and functional properties. The amino-terminal domain spanning about
residues 22 194 of the full-length polypeptide (the N-terminal
methionine is designated residue 1) shows homology to other members
of the tumor necrosis factor receptor (TNFR) family, especially
TNFR-2, through conservation of cysteine rich domains
characteristic of TNFR family members. The carboxy terminal domain
spanning residues 194 401 has no significant homology to any known
sequences. Unlike a number of other TNFR family members, OPG
appears to be exclusively a secreted protein and does not appear to
be synthesized as a membrane associated form.
Based upon its activity as a negative regulator of osteoclast
formation, it is postulated that OPG may bind to a polypeptide
factor involved in osteoclast differentiation and thereby block one
or more terminal steps leading to formation of a mature
osteoclast.
It is therefore an object of the invention to identify polypeptides
which interact with OPG. Said polypeptides may play a role in
osteoclast maturation and may be useful in the treatment of bone
diseases.
SUMMARY OF THE INVENTION
A novel member of the tumor necrosis factor family has been
identified from a murine cDNA library expressed in COS cells
screened using a recombinant OPG-Fc fusion protein as an affinity
probe. The new polypeptide is a transmembrane OPG binding protein
which is predicted to be 316 amino acids in length, and has an
amino terminal cytoplasmic domain, a transmembrane doman, and a
carboxy terminal extracellular domain. OPG binding proteins of the
invention may be membrane-associated or may be in soluble form.
The invention provides for nucleic acids encoding an OPG binding
protein, vectors and host cells expressing the polypeptide, and
method for producing recombinant OPG binding protein. Antibodies or
fragments thereof which specifically bind OPG binding protein are
also provided.
OPG binding proteins may be used in assays to quantitate OPG levels
in biological samples, identify cells and tissues that display OPG
binding protein, and identify new OPG and OPG binding protein
family members. Methods of identifying compounds which interact
with OPG binding protein are also provided. Such compounds include
nucleic acids, peptides, proteins, carbohydrates, lipids or small
molecular weight organic molecules and may act either as agonists
or antagonists of OPG binding protein activity.
OPG binding proteins are involved in osteoclast differentiation and
the level of osteoclast activity in turn modulates bone resorption.
OPG binding protein agonists and antagonists modulate osteoclast
formation and bone resorption and may be used to treat bone
diseases characterized by changes in bone resorption, such as
osteoporosis, hypercalcemia, bone loss due to arthritis metastasis,
immobilization or periodontal disease, Paget's disease,
osteopetrosis, prosthetic loosening and the like. Pharmaceutical
compositions comprising OPG binding proteins and OPG binding
protein agonists and antagonists are also encompassed by the
invention.
DESCRIPTION OF THE FIGURES
FIG. 1. (SEQ ID NO:36 and 37) Structure and sequence of the 32D-F3
insert encoding OPG binding protein. Predicted transmembrane domain
and sites for asparagine-linked carbohydrate chains are
underlined.
FIG. 2. OPG binding protein expression in COS-7 cells transfected
with pcDNA/32D-F3. Cells were lipofected with pcDNA/32D-F3 DNA, the
assayed for binding to either goat anti-human IgG1 alkaline
phosphatase conjugate (secondary alone), human OPG[22-201]-Fc plus
secondary (OPG-Fc), or a chimeric ATAR extracellular domain-Fc
fusion protein (sATAR-Fc). ATAR is a new member of the TNFR
superfamily, and the sATAR-Fc fusion protein serves as a control
for both human IgG1 Fc domain binding, and generic TNFR releated
protein, binding to 32D cell surface molecules.
FIG. 3. Expression of OPG binding protein in human tissues.
Northern blot analysis of human tissue mRNA (Clontech) using a
radiolabeled 32D-F3 derived hybridization probe. Relative molecular
mass is indicated at the left in kilobase pairs (kb). Arrowhead on
right side indicates the migration of an approximately 2.5 kb
transcript detected in lymph node mRNA. A very faint band of the
same mass is also detected in fetal liver.
FIG. 4. (SEQ ID NO:38 and 39) Structure and sequence of the
pcDNA/hu OPGbp 1.1 insert encoding the human OPG binding protein.
The predicted transmembrane domain and site for asparagine-linked
carbohydrate chains are underlined.
FIG. 5. Stimulation of osteoclast development in vitro from bone
marrow macrophage and ST2 cell cocultures treated with recombinant
murine OPG binding protein [158-316]. Cultures were treated with
varying concentrations of murine OPG binding protein ranging from
1.6 to 500 ng/ml . After 8 10 days, cultures were lysed, and TRAP
activity was measured by solution assay. In addition, some cultures
were simultaneously treated with 1 (), 10 (), 100 (), 500 (), and
1000 ng/ml () of recombinant murine OPG [22-401]-Fc protein. Murine
OPG binding protein induces a dose-dependent stimulation in
osteoclast formation, whereas OPG [22-401]-Fc inhibits osteoclast
formation.
FIG. 6. Stimulation of osteoclast development from bone marrow
precursors in vitro in the presence of M-CSF and murine OPG binding
protein [158-316]. Mouse bone marrow was harvested, and cultured in
the presence 250 (), 500 (), 1000 (), and 2000 U/ml () of M-CSF.
Varying concentrations of OPG binding protein [158-316], ranging
from 1.6 to 500 ng/ml, were added to these same cultures.
Osteoclast development was measured by TRAP solution assay.
FIG. 7. Osteoclasts derived from bone marrow cells in the presence
of both M-CSF and OPG binding protein [158-316] resorb bone in
vitro. Bone marrow cells treated with either M-CSF, OPG binding
protein, or with both factors combined, were plated onto bone
slices in culture wells, and were allowed to develop into mature
osteoclasts. The resulting cultures were then stained with
Toluidine Blue (left column), or histochemically to detect TRAP
enzyme activity (right column). In cultures receiving both factors,
mature osteoclasts were formed that were capable of eroding bone as
judged by the presence of blue stained pits on the bone surface.
This correlated with the presence of multiple large,
multinucleated, TRAP positive cells.
FIG. 8. Graph showing the whole blood ionized calcium (iCa) levels
from mice injected with OPG binding protein, 51 hours after the
first injection, and in mice also receiving concurrent OPG
administration. OPG binding protein significantly and dose
dependently increased iCa levels. OPG (1 mg/kg/day) completely
blocked the increase in iCa at a dose of OPG binding protein of 5
ug/day, and partially blocked the increase at a dose of OPG binding
protein of 25 ug/day. (*), different to vehicle treated control
(p<0.05). (#) OPG treated iCa level significantly different to
level in mice receiving that dose of OPG binding protein alone
(p<0.05).
FIG. 9. Radiographs of the left femur and tibia in mice treated
with 0, 5, 25 or 100 ug/day of OPG binding protein for 3.5 days.
There is a dose dependent decrease in bone density evident most
clearly in the proximal tibial metaphysis of these mice, and that
is profound at a dose of 100 ug/day.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides for a polypeptide referred to as an OPG
binding protein, which specifically binds OPG and is involved in
osteoclast differentiation. A cDNA clone encoding the murine form
of the polypeptide was identified from a library prepared from a
mouse myelomonocytic cell line 32-D and transfected into COS cells.
Transfectants were screened for their ability to bind to an
OPG[22-201]-Fc fusion polypeptide (Example 1). The nucleic acid
sequence revealed that OPG binding protein is a novel member of the
TNF family and is most closely related to AGP-1, a polypeptide
previously described in co-owned and co-pending U.S. Ser. No.
08/660,562, filed Jun. 7, 1996, now abandoned. (A polypeptide
identical to AGP-1 and designated TRAIL is described in Wiley et
al. Immunity 3, 673 682 (1995)). OPG binding protein is predicted
to be a type II transmembrane protein having a cytoplamsic domain
at the amino terminus, a transmembrane domain, and a carboxy
terminal extracellular domain (FIG. 1). The amino terminal
cytoplasmic domain spans about residues 1 48, the transmembrane
domain spans about residues 49 69 and the extracellular domain
spans about residues 70 316 as shown in FIG. 1 (SEQ ID NO:37). The
membrane-associated protein specifically binds OPG (FIG. 2). Thus
OPG binding protein and OPG share many characteristics of a
receptor-ligand pair although it is possible that other
naturally-occurring receptors for OPG binding protein exist.
A DNA clone encoding human OPG binding protein was isolated from a
lymph node cDNA library. The human sequence (FIG. 4) is homologous
to the murine sequence. Purified soluble murine OPG binding protein
stimulated osteoclast formation in vitro and induced hypercalcemia
and bone resorption in vivo.
OPG binding protein refers to a polypeptide having an amino acid
sequence of mammalian OPG binding protein, or a fragment, analog,
or derivative thereof, and having at least the activity of binding
OPG. In preferred embodiments, OPG binding protein is of murine or
human origin. In another embodiment, OPG binding protein is a
soluble protein having, in one form, an isolated extracellular
domain separate from cytoplasmic and transmembrane domains. OPG
binding protein is involved in osteoclast differentiation and in
the rate and extent of bone resorption, and was found to stimulate
osteoclast formation and stimulate bone resorption.
Nucleic Acids
The invention provides for isolated nucleic acids encoding OPG
binding proteins. As used herein, the term nucleic acid comprises
cDNA, genomic DNA, wholly or partially synthetic DNA, and RNA. The
nucleic acids of the invention are selected from the group
consisting of:
a) the nucleic acids as shown in FIG. 1 (SEQ ID NO: 36) and FIG. 4
(SEQ ID NO: 38);
b) nucleic acids which hybridize to the polypeptide coding regions
of the nucleic acids shown in FIG. 1 (SEQ ID NO: 36) and FIG. 4
(SEQ ID NO: 38); and remain hybridized to the nucleic acids under
high stringency conditions; and
c) nucleic acids which are degenerate to the nucleic acids of (a)
or (b).
Nucleic acid hybridizations typically involve a multi-step process
comprising a first hybridization step to form nucleic acid duplexes
from single strands followed by a second hybridization step carried
out under more stringent conditions to selectively retain nucleic
acid duplexes having the desired homology. The conditions of the
first hybridization step are generally not crucial, provided they
are not of higher stringency than the second hybridization step.
Generally, the second hybridization is carried out under conditions
of high stringency, wherein "high stringency" conditions refers to
conditions of temperature and salt which are about 12 20.degree. C.
below the melting temperature (T.sub.m) of a perfect hybrid of part
or all of the complementary strands corresponding to FIG. 1 (SEQ.
ID. NO: 36) and FIG. 4 (SEQ ID NO: 38). In one embodiment, "high
stringency" conditions refer to conditions of about 65.degree. C.
and not more than about 1M Na+. It is understood that salt
concentration, temperature and/or length of incubation may be
varied in either the first or second hybridization steps such that
one obtains the hybridizing nucleic acid molecules according to the
invention. Conditions for hybridization of nucleic acids and
calculations of T.sub.m for nucleic acid hybrids are described in
Sambrook et al. Molecular Cloning: A Laboratory Manual Cold Spring
Harbor Laboratory Press, New York (1989).
The nucleic acids of the invention may hybridize to part or all of
the polypeptide coding regions of OPG binding protein as shown in
FIG. 1 (SEQ ID NO: 37) and FIG. 4 (SEQ ID NO: 39); and therefore
may be truncations or extensions of the nucleic acid sequences
shown therein. Truncated or extended nucleic acids are encompassed
by the invention provided that they retain at least the property of
binding OPG. In one embodiment, the nucleic acid will encode a
polypeptide of at least about 10 amino acids. In another
embodiment, the nucleic acid will encode a polypeptide of at least
about 20 amino acids. In yet another embodiment, the nucleic acid
will encode a polypeptide of at least about 50 amino acids. The
hybridizing nucleic acids may also include noncoding sequences
located 5' and/or 3' to the OPG binding protein coding regions.
Noncoding sequences include regulatory regions involved in
expression of OPG binding protein, such as promoters, enhancer
regions, translational initiation sites, transcription termination
sites and the like.
In preferred embodiments, the nucleic acids of the invention encode
mouse or human OPG binding protein. Nucleic acids may encode a
membrane bound form of OPG binding protein or soluble forms which
lack a functional transmembrane region. The predicted transmembrane
region for murine OPG binding protein includes amino acid residues
49 69 inclusive as shown in FIG. 1 (SEQ. ID. NO: 37). The predicted
transmembrane region for human OPG binding protein includes
residues 49 69 as shown in FIG. 4 (SEQ ID NO: 39). Substitutions
which replace hydrophobic amino acid residues in this region with
neutral or hydrophilic amino acid residues would be expected to
disrupt membrane association and result in soluble OPG binding
protein. In addition, deletions of part or all the transmembrane
region would also be expected to produce soluble forms of OPG
binding protein. Nucleic acids encoding amino acid residues 70 316
as shown in FIG. 1 (SEQ ID NO: 37), or fragments and analogs
thereof, encompass soluble OPG binding proteins.
Nucleic acids encoding truncated forms of soluble human OPG binding
proteins are also included. Soluble forms include residues 69 317
as shown in FIG. 4 (SEQ ID NO: 38) and truncations thereof. In one
embodiment, N-terminal truncations generate polypeptides from
residues, 70-317, 71-317, 72-317, and so forth. In another
embodiment, nucleic acids encode soluble OPGbp comprising residues
69 317 and N-terminal truncations thereof up to OPGbp [158-317], or
alternatively, up to OPGbp [166-317].
Plasmid phuOPGbp 1.1 in E. coli strain DH10 encoding human OPG
binding protein was deposited with the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 on
Jun. 13, 1997.
Nucleic acid sequences of the invention may be used for the
detection of sequences encoding OPG binding protein in biological
samples. In particular, the sequences may be used to screen cDNA
and genomic libraries for related OPG binding protein sequences,
especially those from other species. The nucleic acids are also
useful for modulating levels of OPG binding protein by anti-sense
technology or in vivo gene expression. Development of transgenic
animals expressing OPG binding protein is useful for production of
the polypeptide and for the study of in vivo biological
activity.
Vectors and Host Cells
The nucleic acids of the invention will be linked with DNA
sequences so as to express biologically active OPG binding protein.
Sequences required for expression are known to those skilled in the
art and include promoters and enhancer sequences for initiation of
RNA synthesis, transcription termination sites, ribosome binding
sites for the initiation of protein synthesis, and leader sequences
for secretion. Sequences directing expression and secretion of OPG
binding protein may be homologous, i.e., the sequences are
identical or similar to those sequences in the genome involved in
OPG binding protein expression and secretion, or they may be
heterologous. A variety of plasmid vectors are available for
expressing OPG binding protein in host cells (see, for example,
Methods in Enzymology v. 185, Goeddel, D. V. ed., Academic Press
(1990)). For expression in mammalian host cells, a preferred
embodiment is plasmid pDSR.alpha. described in PCT Application No.
90/14363. For expression in bacterial host cells, preferred
embodiments include plasmids harboring the lux promoter (see
co-owned and co-pending U.S. Ser. No. 08/577,778, filed Dec. 22,
1995). In addition, vectors are available for the tissue-specific
expression of OPG binding protein in transgenic animals. Retroviral
and adenovirus-based gene transfer vectors may also be used for the
expression of OPG binding protein in human cells for in vivo
therapy (see PCT Application No. 86/00922).
Procaryotic and eucaryotic host cells expressing OPG binding
protein are also provided by the invention. Host cells include
bacterial, yeast, plant, insect or mammalian cells. OPG binding
protein may also be produced in transgenic animals such as mice or
goats. Plasmids and vectors containing the nucleic acids of the
invention are introduced into appropriate host cells using
transfection or transformation techniques known to one skilled in
the art. Host cells may contain DNA sequences encoding OPG binding
protein as shown in FIG. 1 or a portion thereof, such as the
extracellular domain or the cytoplasmic domain. Nucleic acids
encoding OPG binding proteins may be modified by substitution of
codons which allow for optimal expression in a given host. At least
some of the codons may be so-called preference codons which do not
alter the amino acid sequence and are frequently found in genes
that are highly expressed. However, it is understood that codon
alterations to optimize expression are not restricted to the
introduction of preference codons. Examples of preferred mammalian
host cells for OPG binding protein expression include, but are not
limited to COS, CHOd-, 293 and 3T3 cells. A preferred bacterial
host cell is Escherichia coli.
Polypeptides
The invention also provides OPG binding protein as the product of
procaryotic or eucaryotic expression of an exogenous DNA sequence,
i.e., OPG binding protein is recombinant OPG binding protein.
Exogenous DNA sequences include cDNA, genomic DNA and synthetic DNA
sequences. OPG binding protein may be the product of bacterial,
yeast, plant, insect or mammalian cells expression, or from
cell-free translation systems. OPG binding protein produced in
bacterial cells will have an N-terminal methionine residue. The
invention also provides for a process of producing OPG binding
protein comprising growing procaryotic or eucaryotic host cells
transformed or transfected with nucleic acids encoding OPG binding
protein and isolating polypeptide expression products of the
nucleic acids.
Polypeptides which are mamalian OPG binding proteins or are
fragments, analogs or derivatives thereof are encompassed by the
invention. In a preferred embodiment, the OPG binding protein is
human OPG binding protein. A fragment of OPG binding protein refers
to a polypeptide having a deletion of one or more amino acids such
that the resulting polypeptide has at least the property of binding
OPG. Said fragments will have deletions originating from the amino
terminal end, the carboxy terminal end, and internal regions of the
polypeptide. Fragments of OPG binding protein are at least about
ten amino acids, at least about 20 amino acids, or at least about
50 amino acids in length. In preferred embodiments, OPG binding
protein will have a deletion of one or more amino acids from the
transmembrane region (amino acid residues 49 69 as shown in FIG.
1), or, alternatively, one or more amino acids from the
amino-terminus up to and/or including the transmembrane region
(amino acid residues 1 49 as shown in FIG. 1). In another
embodiment, OPG binding protein is a soluble protein comprising,
for example, amino acid residues 69 316, or 70 316, or N-terminal
or C-terminal truncated forms thereof, which retain OPG binding
activity. OPG binding protein is also a human soluble protein as
shown in FIG. 4 comprising residues 69 317 as shown in FIG. 4 and
N-terminal truncated forms thereof, e.g., 70-517, 71-517, 71-317,
72-317 and so forth. In a preferred embodiment, the soluble human
OPG binding protein comprising residues 69 317 and N-terminal
truncation thereof up to OPGbp [158-317], or alternatively up to
OPG [166-317].
An analog of an OPG binding protein refers to a polypeptide having
a substitution or addition of one or more amino acids such that the
resulting polypeptide has at least the property of binding OPG.
Said analogs will have substitutions or additions at any place
along the polypeptide. Preferred analogs include those of soluble
OPG binding proteins. Fragments or analogs may be naturally
occurring, such as a polypeptide product of an allelic variant or a
mRNA splice variant, or they may be constructed using techniques
available to one skilled in the art for manipulating and
synthesizing nucleic acids. The polypeptides may or may not have an
amino terminal methionine residue
Also included in the invention are derivatives of OPG binding
protein which are polypeptides that have undergone
post-translational modifications (e.g., addition of N-linked or
O-linked carbohydrate chains, processing of N-terminal or
C-terminal ends), attachment of chemical moieties to the amino acid
backbone, chemical modifications of N-linked or O-linked
carbohydrate chains, and addition of an N-terminal methionine
residue as a result of procaryotic host cell expression. In
particular, chemically modified derivatives of OPG binding protein
which provide additional advantages such as increased stability,
longer circulating time, or decreased immunogenicity are
contemplated. Of particular use is modification with water soluble
polymers, such as polyethylene glycol and derivatives thereof (see
for example U.S. Pat. No. 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
Polypeptides may also be modified at pre-determined positions in
the polypeptide, such as at the amino terminus, or at a selected
lysine or arginine residue within the polypeptide. Other chemical
modificaitons provided include a detectable label, such as an
enzymatic, fluorescent, isotopic or affinity label to allow for
detection and isolation of the protein.
OPG binding protein chimeras comprising part or all of an OPG
binding protein amino acid sequence fused to a heterologous amino
acid sequence are also included. The heterologous sequence may be
any sequence which allows the resulting fusion protein to retain
the at least the activity of binding OPG. In a preferred
embodiment, the carboxy terminal extracellular domain of OPG
binding protein is fused to a heterologous sequence. Such sequences
include heterologous cytoplasmic domains that allow for alternative
intracellular signalling events, sequences which promote
oligomerization such as the Fc region of IgG, enzyme sequences
which provide a label for the polypeptide, and sequences which
provide affinity probes, such as an antigen-antibody
recognition.
The polypeptides of the invention are isolated and purified from
tissues and cell lines which express OPG binding protein, either
extracted from lysates or from conditioned growth medium, and from
transformed host cells expressing OPG binding protein. OPG binding
protein may be obtained from murine myelomonocytic cell line 32-D
(ATCC accession no. CRL-11346). Human OPG binding protein, or
nucleic acids encoding same, may be isolated from human lymph node
or fetal liver tissue. Isolated OPG binding protein is free from
association with human proteins and other cell constituents.
A method for the purification of OPG binding protein from natural
sources (e.g. tissues and cell lines which normally express OPG
binding protein) and from transfected host cells is also
encompassed by the invention. The purification process may employ
one or more standard protein purification steps in an appropriate
order to obtain purified protein. The chromatography steps can
include ion exchange, gel filtration, hydrophobic interaction,
reverse phase, chromatofocusing, affinity chromatography employing
an anti-OPG binding protein antibody or biotin-streptavidin
affinity complex and the like.
Antibodies
Antibodies specifically binding the polypeptides of the invention
are also encompassed by the invention. The antibodies may be
produced by immunization with full-length OPG binding protein,
soluble forms of OPG binding protein, or a fragment thereof. The
antibodies of the invention may be polyclonal or monoclonal, or may
be recombinant antibodies, such as chimeric antibodies wherein the
murine constant regions on light and heavy chains are replaced by
human sequences, or CDR-grafted antibodies wherein only the
complementary determining regions are of murine origin. Antibodies
of the invention may also be human antibodies prepared, for
example, by immunization of transgenic animals capable of producing
human antibodies (see, for example, PCT Application No.
WO93/12227). The antibodies are useful for detecting OPG binding
protein in biological samples, thereby allowing the identification
of cells or tissues which produce the protein In addition,
antibodies which bind to OPG binding protein and block interaction
with other binding compounds may have therapeutic use in modulating
osteoclast differentiation and bone resorption.
Antibodies to the OPG binding protein may be useful in treatment of
bone diseases such as, osteoporosis and Paget's disease. Antibodies
can be tested for binding to the OPG binding protein in the absence
or presence of OPG and examined for their ability to inhibit ligand
(OPG binding protein) mediated osteoclastogenesis and/or bone
resorption. It is also anticipated that the peptides themselves may
act as an antagonist of the ligand:receptor interaction and inhibit
ligand-mediated osteoclastogenesis, and peptides of the OPG binding
protein will be explored for this purpose as well.
Compositions
The invention also provides for pharmaceutical compositions
comprising a therapeutically effective amount of the OPG binding
protein of the invention together with a pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative
and/or adjuvant. The invention also provides for pharmaceutical
compositions comprising a therapeutically effective amount of an
OPG binding protein agonist or antagonist. The term
"therapeutically effective amount" means an amount which provides a
therapeutic effect for a specified condition and route of
administration. The composition may be in a liquid or lyophilized
form and comprises a diluent (Tris, acetate or phosphate buffers)
having various pH values and ionic strengths, solubilizer such as
Tween or Polysorbate, carriers such as human serum albumin or
gelatin, preservatives such as thimerosal or benzyl alcohol, and
antioxidants such as ascrobic acid or sodium metabisulfite.
Selection of a particular composition will depend upon a number of
factors, including the condition being treated, the route of
administration and the pharmacokinetic parameters desired. A more
extensive survey of component suitable for pharmaceutical
compositions is found in Remington's Pharmaceutical Sciences, 18th
ed. A. R. Gennaro, ed. Mack, Easton, Pa. (1980).
In a preferred embodiment, compositions comprising soluble OPG
binding proteins are also provided. Also encompassed are
compositions comprising soluble OPG binding protein modified with
water soluble polymers to increase solubility, stability, plasma
half-life and bioavailability. Compositions may also comprise
incorporation of soluble OPG binding protein into liposomes,
microemulsions, micelles or vesicles for controlled delivery over
an extended period of time. Soluble OPG binding protein may be
formulated into microparticles suitable for pulmonary
administration.
Compositions of the invention may be administered by injection,
either subcutaneous, intravenous or intramuscular, or by oral,
nasal, pulmonary or rectal administration. The route of
administration eventually chosen will depend upon a number of
factors and may be ascertained by one skilled in the art.
The invention also provides for pharmaceutical compositions
comprising a therapeutically effective amount of the nucleic acids
of the invention together with a pharmaceutically acceptable
adjuvant. Nucleic acid compositions will be suitable for the
delivery of part or all of the coding region of OPG binding protein
and/or flanking regions to cells and tissues as part of an
anti-sense therapy regimen.
Methods of Use
OPG binding proteins may be used in a variety of assays for
detecting OPG and characterizing interactions with OPG. In general,
the assay comprises incubating OPG binding protein with a
biological sample containing OPG under conditions which permit
binding to OPG to OPG binding protein, and measuring the extent of
binding. OPG may be purified or present in mixtures, such as in
body fluids or culture medium. Assays may be developed which are
qualitative or quantitative, with the latter being useful for
determining the binding parameters (affinity constants and
kinetics) of OPG to OPG binding protein and for quantitating levels
of biologically active OPG in mixtures. Assays may also be used to
evaluate the binding of OPG to fragments, analogs and derivatives
of OPG binding protein and to identify new OPG and OPG binding
protein family members.
Binding of OPG to OPG binding protein may be carried out in several
formats, including cell-based binding assays, membrane binding
assays, solution-phase assays and immunoassays. In general, trace
levels of labeled OPG are incubated with OPG binding protein
samples for a specified period of time followed by measurement of
bound OPG by filtration, electrochemiluminescent (ECL, ORIGEN
system by IGEN), cell-based or immunoassays. Homogeneous assay
technologies for radioactivity (SPA; Amersham) and time resolved
fluoresence (HTRF, Packard) can also be implemented. Binding is
detected by labeling OPG or an anti-OPG antibody with radioactive
isotopes (125I, 35S, 3H), fluorescent dyes (fluorescein),
lanthanide (Eu3+) chelates or cryptates, orbipyridyl-ruthenium
(Ru2+) complexes. It is understood that the choice of a labeled
probe will depend upon the detection system used. Alternatively,
OPG may be modified with an unlabled epitope tag (e.g., biotin,
peptides, His.sub.6, myc) and bound to proteins such as
streptavidin, anti-peptide or anti-protein antibodies which have a
detectable label as described above.
In an alternative method, OPG binding protein may be assayed
directly using polyclonal or monoclonal antibodies to OPG binding
proteins in an immunoassay. Additional forms of OPG binding
proteins containing epitope tags as described above may be used in
solution and immunoassays.
Methods for indentifying compounds which interact with OPG binding
protein are also encompassed by the invention. The method comprises
incubating OPG binding protein with a compound under conditions
which permit binding of the compound to OPG binding protein, and
measuring the extent of binding. The compound may be substantially
purified or present in a crude mixture. Binding compounds may be
nucleic acids, proteins, peptides, carbohydrates, lipids or small
molecular weight organic compounds. The compounds may be further
characterized by their ability to increase or decrease OPG binding
protein activity in order to determine whether they act as an
agonist or an antagonist.
OPG binding proteins are also useful for identification of
intracellular proteins which interact with the cytoplasmic domain
by a yeast two-hybrid screening process. As an example, hybrid
constructs comprising DNA encoding the N-terminal 50 amino acids of
an OPG binding protein fused to a yeast GAL4-DNA binding domain may
be used as a two-hybrid bait plasmid. Positive clones emerging from
the screening may be characterized further to identify interacting
proteins. This information may help elucidate a intracellular
signaling mechanism associated with OPG binding protein and provide
intracellular targets for new drugs that modulate bone
resorption.
OPG binding protein may be used to treat conditions characterized
by excessive bone density. The most common condition is
osteopetrosis in which a genetic defect results in elevated bone
mass and is usually fatal in the first few years of life.
Osteopetrosis is preferably treated by administration of soluble
OPG binding protein.
The invention also encompasses modulators (agonists and
antagonists) of OPG binding protein and the methods for obtaining
them. An OPG binding protein modulator may either increase or
decrease at least one activity associated with OPG binding protein,
such as ability to bind OPG or some other interacting molecule or
to regulate osteoclast maturation. Typically, an agonist or
antagonist may be a co-factor, such as a protein, peptide,
carbohydrate, lipid or small molecular weight molecule, which
interacts with OPG binding protein to regulate its activity.
Potential polypeptide antagonists include antibodies which react
with either soluble or membrane-associated forms of OPG binding
protein, and soluble forms of OPG binding protein which comprise
part or all of the extracellular domain of OPG binding protein.
Molecules which regulate OPG binding protein expression typically
include nucleic acids which are complementary to nucleic acids
encoding OPG binding protein and which act as anti-sense regulators
of expression.
OPG binding protein is involved in controlling formation of mature
osteoclasts, the primary cell type implicated in bone resorption.
An increase in the rate of bone resorption (over that of bone
formation) can lead to various bone disorders collectively referred
to as osteopenias, and include osteoporosis, osteomyelitis,
hypercalcemia, osteopenia brought on by surgery or steroid
administration, Paget's disease, osteonecrosis, bone loss due to
rheumatoid arthritis, periodontal bone loss, immobilization,
prosthetic loosing and osteolytic metastasis. Conversely, a
decrease in the rate of bone resorption can lead to osteopetrosis,
a condition marked by excessive bone density. Agonists and
antagonists of OPG binding protein modulate osteoclast formation
and may be administered to patients suffering from bone disorders.
Agonists and antagonists of OPG binding protein used for the
treatment of osteopenias may be administered alone or in
combination with a therapeutically effective amount of a bone
growth promoting agent including bone morphogenic factors
designated BMP-1 to BMP-12, transforming growth factor-.beta. and
TGF-.beta. family members, fibroblast growth factors FGF-1 to
FGF-10, interleukin-1 inhibitors, TNF.alpha. inhibitors,
parathyroid hormone, E series prostaglandins, bisphosphonates and
bone-enhancing minerals such as fluoride and calcium. Antagonists
of OPG binding proteins may be particularly useful in the treatment
of osteopenia.
The following examples are offered to more fully illustrate the
invention, but are not construed as limiting the scope thereof.
EXAMPLE 1
Identification of a Cell Line Source for an OPG Binding Protein
Osteoprotegerin (OPG) negatively regulates osteoclastogenesis in
vitro and in vivo. Since OPG is a TNFR-related protein, it is
likely to interact with a TNF-related family member while mediating
its effects. With one exception, all known members of the TNF
superfamily are type II transmembrane proteins expressed on the
cell surface. To identify a source of an OPG binding protein,
recombinant OPG-Fc fusion proteins were used as immunoprobes to
screen for OPG binding proteins located on the surface of various
cell lines and primary hematopoietic cells.
Cell lines that grew as adherent cultures in vitro were treated
using the following methods: Cells were plated into 24 well tissue
culture plates (Falcon), then allowed to grow to approximately 80%
confluency. The growth media was then removed, and the adherent
cultures were washed with phosphate buffered saline (PBS) (Gibco)
containing 1% fetal calf serum (FCS). Recombinant mouse OPG
[22-194]-Fc and human OPG [22-201]-Fc fusion proteins (see U.S.
Ser. No. 08/706,945 filed Sep. 3, 1996 now U.S. Pat. No. 6,369,027)
were individually diluted to 5 ug/ml in PBS containing 1% FCS, then
added to the cultures and allowed to incubate for 45 min at
0.degree. C. The OPG-Fc fusion protein solution was discarded, and
the cells were washed in PBS-FCS solution as described above. The
cultures were then exposed to phycoeyrthrin-conguated goat F(ab')
anti-human IgG secondary antibody (Southern Biotechnology
Associates Cat. # 2043-09) diluted into PBS-FCS. After a 30 45 min
incubation at 0.degree. C., the solution was discarded, and the
cultures were washed as described above. The cells were then
analysed by immunofluorescent microscopy to detect cell lines which
express a cell surface OPG binding protein.
Suspension cell cultures were analysed in a similar manner with the
following modifications: The diluent and wash buffer consisted of
calcium- and magnesium-free phosphate buffered saline containing 1%
FCS. Cells were harvested from exponentially replicating cultures
in growth media, pelleted by centrifugation, then resuspended at
1.times.10.sup.7 cells/ml in a 96 well microtiter tissue culture
plate (Falcon). Cells were sequentially exposed to recombinant
OPG-Fc fusion proteins, then secondary antibody as described above,
and the cells were washed by centrifugation between each step. The
cells were then analysed by fluorescence activated cell sorting
(FACS) using a Becton Dickinson FACscan.
Using this approach, the murine myelomonocytic cell line 32D (ATCC
accession no. CRL-11346) was found to express a surface molecule
which could be detected with both the mouse OPG[22-194]-Fc and the
human OPG[22-201]-Fc fusion proteins. Secondary antibody alone did
not bind to the surface of 32D cells nor did purified human IgG1
Fc, indicating that binding of the OPG-Fc fusion proteins was due
to the OPG moiety. This binding could be competed in a dose
dependent manner by the addition of recombinant murine or human
OPG[22-401] protein. Thus the OPG region required for its
biological activity is capable of specifically binding to a
32D-derived surface molecule.
EXAMPLE 2
Expression Cloning of a Murine OPG Binding Protein
A cDNA library was prepared from 32D mRNA, and ligated into the
mammalian expression vector pcDNA3.1(+) (Invitrogen, San Diego,
Calif.). Exponentially growing 32D cells maintained in the presence
of recombinant interleukin-3 were harvested, and total cell RNA was
purified by acid guanidinium thiocyanate-phenol-chloroform
extraction (Chomczynski and Sacchi. Anal. Biochem. 162, 156 159,
(1987)). The poly (A+) mRNA fraction was obtained from the total
RNA preparation by adsorption to, and elution from, Dynabeads Oligo
(dT)25 (Dynal Corp) using the manufacturer's recommended
procedures. A directional, oligo-dT primed cDNA library was
prepared using the Superscript Plasmid System (Gibco BRL,
Gaithersburg, Md.) using the manufacturer's recommended procedures.
The resulting cDNA was digested to completion with Sal I and Not I
restriction endonuclease, then fractionated by size exclusion gel
chromatography. The highest molecular weight fractions were
selected, and then ligated into the polyliker region of the plasmid
vector pcDNA3.1(+) (Invitrogen, San Diego, Calif.). This vector
contains the CMV promotor upstream of multiple cloning site, and
directs high level expression in eukaryotic cells. The library was
then electroporated into competent E. coli (ElectroMAX DH10B,
Gibco, N.Y.), and titered on LB agar containing 100 ug/ml
ampicillin. The library was then arrayed into segregated pools
containing approximately 1000 clones/pool, and 1.0 ml cultures of
each pool were grown for 16 20 hr at 37.degree. C. Plasmid DNA from
each culture was prepared using the Qiagen Qiawell 96 Ultra Plasmid
Kit (catalog #16191) following manufacturer's recommended
procedures.
Arrayed pools of 32D cDNA expression library were individually
lipofected into COS-7 cultures, then assayed for the acquisition of
a cell surface OPG binding protein. To do this, COS-7 cells were
plated at a density of 1.times.10.sup.6 per ml in six-well tissue
culture plates (Costar), then cultured overnight in DMEM (Gibco)
containing 10% FCS. Approximately 2 .mu.g of plasmid DNA from each
pool was diluted into 0.5 ml of serum-free DMEM, then sterilized by
centrifugation through a 0.2 .mu.m Spin-X column (Costar).
Simultaneously, 10 .mu.l of Lipofectamine (Life Technologies Cat #
18324-012) was added to a separate tube containing 0.5 ml of
serum-free DMEM. The DNA and Lipofectamine solutions were mixed,
and allowed to incubate at RT for 30 min. The COS-7 cell cultures
were then washed with serum-free DMEM, and the DNA-lipofectamine
complexes were exposed to the cultures for 2 5 hr at 37.degree. C.
After this period, the media was removed, and replaced with DMEM
containing 10% FCS. The cells were then cultured for 48 hr at
37.degree. C.
To detect cultures that express an OPG binding protein, the growth
media was removed, and the cells were washed with PBS-FCS solution.
A 1.0 ml volume of PBS-FCS containing 5 .mu.g/ml of human
OPG[22-201]-Fc fusion protein was added to each well and incubated
at RT for 1 hr. The cells were washed three times with PBS-FCS
solution, and then fixed in PBS containing 2% paraformaldehyde and
0.2% glutaraldehyde in PBS at RT for 5 min. The cultures were
washed once with PBS-FCS, then incubated for 1 hr at 65.degree. C.
while immersed in PBS-FCS solution. The cultures were allowed to
cool, and the PBS-FCS solution was aspirated. The cultures were
then incubated with an alkaline-phosphatase conjugated goat
anti-human IgG (Fc specific) antibody (SIGMA Product # A-9544) at
Rt for 30 min, then washed three-times with 20 mM Tris-Cl (pH 7.6),
and 137 mM NaCl. Immune complexes that formed during these steps
were detected by assaying for alkaline phosphatase activity using
the Fast Red TR/AS-MX Substrate Kit (Pierce, Cat. # 34034)
following the manufacturer's recommended procedures.
Using this approach, a total of approximately 300,000 independent
32D cDNA clones were screened, represented by 300 transfected pools
of 1000 clones each. A single well was identifed that contained
cells which acquired the ability to be specifically decorated by
the OPG-Fc fusion protein. This pool was subdivided by sequential
rounds of sib selection, yeilding a single plasmid clone 32D-F3
(FIG. 1). 32D-F3 plasmid DNA was then transfected into COS-7 cells,
which were immunostained with either FITC-conjugated goat
anti-human IgG secondary antibody alone, human OPG[22-201]-Fc
fusion protein plus secondary, or with ATAR-Fc fusion protein (ATAR
also known as HVEM; Montgomery et al. Cell 87, 427 436 (1996))
(FIG. 2). The secondary antibody alone did not bind to COS-7/32D-F3
cells, nor did the ATAR-Fc fusion protein. Only the OPG Fc fusion
protein bound to the COS-7/32D-F3 cells, indicating that 32D-F3
encoded an OPG binding protein displayed on the surface of
expressing cells.
EXAMPLE 3
OPG Binding Protein Sequence
The 32D-F3 clone isolated above contained an approximately 2.3 kb
cDNA insert (FIG. 1), which was sequenced in both directions on an
Applied Biosystems 373A automated DNA sequencer using primer-driven
Taq dye-terminator reactions (Applied Biosystems) following the
manufacturer's recommended procedures. The resulting nucleotide
sequence obtained was compared to the DNA sequence database using
the FASTA program (GCG, Univeristy of Wisconsin), and analysed for
the presence of long open reading frames (LORF's) using the
"Six-way open reading frame" application (Frames) (GCG, Univeristy
of Wisconsin). A LORF of 316 amino acid (aa) residues beginning at
methionine was detected in the appropriate orientation, and was
preceded by a 5' untranslated region of about 150 bp. The 5'
untranslated region contained an in-frame stop codon upstream of
the predicted start codon. This indicates that the structure of the
32D-F3 plasmid is consistent with its ability to utilize the CMV
promotor region to direct expression of a 316 aa gene product in
mammalian cells.
The predicted OPG binding protein sequence was then compared to the
existing database of known protein sequences using a modified
version of the FASTA program (Pearson, Meth. Enzymol. 183, 63 98
(1990)). The amino acid sequence was also analysed for the presence
of specific motifs conserved in all known members of the tumor
necrosis factor (TNF) superfamily using the sequence profile method
of (Gribskov et al. Proc. Natl. Acad. Sci. USA 83, 4355 4359
(1987)), as modified by Luethy et al. Protein Sci. 3, 139 146
(1994)). There appeared to be significant homology throughout the
OPG binding protein to several members of the TNF superfamily. The
mouse OPG binding protein appear to be most closely related to the
mouse and human homologs of both TRAIL and CD40 ligand. Further
analysis of the OPG binding protein sequence indicated a strong
match to the TNF superfamily, with a highly significant Z score of
19.46.
The OPG binding protein amino acid sequence contains a probable
hydrophobic transmembrane domain that begins at a M49 and extends
to L69. Based on this configuration relative to the methionine
start codon, the OPG binding protein is predicted to be a type II
transmembrane protein, with a short N-terminal intracellular
domain, and a longer C-terminal extracellular domain (FIG. 4). This
would be similar to all known TNF family members, with the
exception of lymphotoxin alpha (Nagata and Golstein, Science 267,
1449 1456 (1995)).
EXAMPLE 4
Expression of Human OPG Binding Protein mRNA
Multiple human tissue northern blots (Clontech, Palo Alto, Calif.)
were probed with a .sup.32P-dCTP labelled 32D-F3 restriction
fragment to detect the size of the human transcript and to
determine patterns of expression. Northern blots were prehybridized
in 5.times.SSPE, 50% formamide, 5.times. Denhardt's solution, 0.5%
SDS, and 100 .mu.g/ml denatured salmon sperm DNA for 2 4 hr at
42.degree. C. The blots were then hybridized in 5.times.SSPE, 50%
formamide, 2.times. Denhardt's solution, 0.1% SDS, 100 .mu.g/ml
denatured salmon sperm DNA, and 5 ng/ml labelled probe for 18 24 hr
at 42.degree. C. The blots were then washed in 2.times.SSC for 10
min at RT, 1.times.SSC for 10 min at 50.degree. C., then in
0.5.times.SSC for 10 15 min.
Using a probe derived from the mouse cDNA and hybridization under
stringent conditions, a predominant mRNA species with a relative
molecular mass of about 2.5 kb was detected in lymph nodes (FIG.
3). A faint signal was also detected at the same relative molecular
mass in fetal liver mRNA. No OPG binding protein transcripts were
detected in the other tissues examined. The data suggest that
expression of OPG binding protein mRNA was extremely restricted in
human tissues. The data also indicate that the cDNA clone isolated
is very close to the size of the native transcript, suggesting
32D-F3 is a full length clone.
EXAMPLE 5
Molecular Cloning of the Human OPG Binding Protein
The human homolog of the OPG binding protein is expressed as an
approximately 2.5 kb mRNA in human peripheral lymph nodes and is
detected by hybridization with a mouse cDNA probe under stringent
hybdization conditions. DNA encoding human OPG binding protein is
obtained by screening a human lymph node cDNA library by either
recombinant bacteriphage plaque, or transformed bacterial colony,
hybridiziation methods (Sambrook et al. Molecular Cloning: A
Laboratory Manual Cold Spring Harbor Press, New York (1989)). To
this the phage or plasmid cDNA library are screened using
radioactively-labeled probes derived from the murine OPG binding
protein clone 32D-F3. The probes are used to screen nitrocellulose
filter lifted from a plated library. These filters are
prehybridized and then hybridized using conditions specified in
Example 4, ultimately giving rise to purified clones of the human
OPG binding protein cDNA. Inserts obtained from any human OPG
binding protein clones would be sequenced and analysed as described
in Example 3.
A human lymph node poly A+ RNA (Clontech, Inc., Palo Alto, Calif.)
was analysed for the presence of OPG-bp transcripts as previously
in U.S. Ser. No. 08/577,788, filed Dec. 22, 1995. A northern blot
of this RNA sample probed under stringent conditions with a
32P-labeled mouse OPG-bp probe indicated the presence of human
OPG-bp transcripts. An oligo dT-primed cDNA library was then
synthesized from the lymph node mRNA using the SuperScript kit
(GIBCO life Technologies, Gaithersberg, Md.) as described in
example 2. The resulting cDNA was size selected, and the high
molecular fraction ligated to plasmid vector pcDNA 3.1 (+)
(Invitrogen, San Diego, Calif.). Electrocompetent E. coli DH10
(GIBCO life Technologies, Gaithersberg, Md.) were transformed, and
1.times.10.sup.6 ampicillin resistant transformants were screened
by colony hybridization using a 32P-labeled mouse OPG binding
protein probe.
A plasmid clone of putative human OPG binding protein cDNA was
isolated, phuOPGbp-1.1, and contained a 2.3 kp insert. The
resulting nucleotide sequence of the phuOPGbp-1.1 insert was
approximately 80 85% homologous to the mouse OPG binding protein
cDNA sequence. Translation of the insert DNA sequence indicated the
presence of a long open reading frame predicted to encode a 317 aa
polypeptide (FIG. 4). Comparison of the mouse and human OPG-bp
polypeptides shows that they are .about.87% identical, indicating
that this protein is highly conserved during evolution.
The human OPG binding protein DNA and protein sequences were not
present in Genbank, and there were no homologus EST sequences. As
with the murine homolog, the human OPG binding protein shows strong
sequence similarity to all members of the TNF.alpha. superfamily of
cytokines.
EXAMPLE 6
Cloning and Bacterial Expression of OPG Binding Protein
PCR amplification employing the primer pairs and templates
described below are used to generate various forms of murine OPG
binding proteins. One primer of each pair introduces a TAA stop
codon and a unique XhoI or SacII site following the carboxy
terminus of the gene. The other primer of each pair introduces a
unique NdeI site, a N-terminal methionine, and optimized codons for
the amino terminal portion of the gene. PCR and thermocycling is
performed using standard recombinant DNA methodology. The PCR
products are purified, restriction digested, and inserted into the
unique NdeI and XhoI or SacII sites of vector pAMG21 (ATCC
accession no. 98113) and transformed into the prototrophic E. coli
393 or 2596. Other commonly used E. coli expression vectors and
host cells are also suitable for expression. After transformation,
the clones are selected, plasmid DNA is isolated and the sequence
of the OPG binding protein insert is confirmed.
pAMG21-Murine OPG Binding Protein [75-316]
This construct was engineered to be 242 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met(75)-Asp-Pro-Asn-Arg-------Gln-Asp-Ile-Asp(316)-COOH
(SEQ ID NO: 1). The template to be used for PCR was pcDNA/32D-F3
and oligonucleotides #1581-72 and #1581-76 were the primer pair to
be used for PCR and cloning this gene construct.
TABLE-US-00001 1581-72:
5'-GTTCTCCTCATATGGATCCAAACCGTATTTCTGAAGACAGCACTCACTGCTT-3' (SEQ ID
NO:2) 1581-76: 5'-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID
NO:3)
pAMG21-Murine OPG Binding Protein [95-316]
This construct was engineered to be 223 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met-His(95)-Glu-Asn-Ala-Gly-------Gln-Asp-Ile-Asp(316)--COOH
(SEQ ID NO: 2). The template used for PCR was pcDNA/32D-F3 and
oligonucleotides #1591-90 and #1591-95 were the primer pair used
for PCR and cloning this gene construct.
TABLE-US-00002 1591-90:
5'-ATTTGATTCTAGAAGGAGGAATAACATATCCATGAAAACGCAGGTCTGCAG-3' (SEQ ID
NO:5) 1591-95: 5'-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3'
(SEQ ID NO:5)
pAMG21-Murine OPG Binding Protein [107-316]
This construct was engineered to be 211 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met-Ser(107)-Glu-Asp-Thr-Leu-------Gln-Asp-Ile-Asp(316)-COOH
(SEQ ID NO: 7). The template used for PCR was pcDNA/32D-F3 and
oligonucleotides #1591-93 and #1591-95 were the primer pair used
for PCR and cloning this gene construct.
TABLE-US-00003 1591-93:
5'-ATTTGATTCTAGAAGGAGGAATAACATATGTCTGAAGACACTCTGCCGGACTCC-3' (SEQ
ID NO:8) 1591-95: 5'-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3'
(SEQ ID NO:6)
pAMG21-Murine OPG Binding Protein [118-316]
This construct was engineered to be 199 amino acids in length and
have the following N-terminal and C-terminal residues, NH.sub.2-Met
(118)-Lys-Gln-Ala-Phe-Gln-------Gln-Asp-Ile-Asp(316)-COOH (SEQ ID
NO: 9). The template used for PCR was pcDNA/32D-F3 and
oligonucleotides #1591-94 and #1591-95 were the primer pair used
for PCR and cloning this gene construct.
TABLE-US-00004 1591-94:
5'-ATTTGATTCTAGAAGGAGGAATAACATATGAAACAAGCTTTTCAGGGG-3' (SEQ ID
NO:10) 1591-95: 5'-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3'
(SEQ ID NO:6)
pAMG21-Murine OPG Binding Protein [128-316]
This construct was engineered to be 190 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met-Lys(128)-Glu-Leu-Gln-His-------Gln-Asp-Ile-Asp(316)-COOH
(SEQ ID NO: 11). The template used for PCR was pcDNA/32D-F3 and
oligonucleotides #1591-91 and #1591-95 were the primer pair used
for PCR and cloning this gene construct.
TABLE-US-00005 1591-91:
5'-ATTTGATTCTAGAAGGAGGAATAACATATGAAAGAACTGCAGCACATTGTG-3' (SEQ ID
NO:12) 1591-95: 5'-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3'
(SEQ ID NO:6)
pAMG2'-Murine OPG Binding Protein [137-316]
This construct was engineered to be 181 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met-Gln(137)-Arg-Phe-Ser-Gly-------Gln-Asp-Ile-Asp(316)--COOH
(SEQ ID NO: 13). The template used for PCR was pcDNA/32D-F3 and
oligonucleotides #1591-92 and #1591-95 were the primer pair used
for PCR and cloning this gene construct.
TABLE-US-00006 1591-92:
5'-ATTTGATTCTAGAAGGAGGAATAACATATGCAGCGTTTCTCTGGTGCTCCA-3' (SEQ ID
NO:14) 1591-95: 5'-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3'
(SEQ ID NO:6)
pAMG21-Murine OPG Binding Protein [146-316]
This construct is engineered to be 171 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met(146)-(Gly-Ser-Trp--------Gln-Asp-Ile-Asp(316)-COOH
(SEQ ID NO: 15). The template to be used for PCR is pAMG21-murine
OPG binding protein [75-316] described above and oligonucleotides
#1600-98 and #1581-76 will be the primer pair to be used for PCR
and cloning this gene construct.
TABLE-US-00007 1600-98:
5'-GTTCTCCTCATATGGAAGGTTCTTGGTTGGATGTGGCCCA-3' (SEQ ID NO:16)
1581-76: 5'-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID
NO:3)
pAMG21-Murine OPG Binding Protein [156-316]
This construct is engineered to be 162 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met-Arg(156)-Gly-Lys-Pro--------Gln-Asp-Ile-Asp (316)-COOH
(SEQ ID NO: 17). The template to be used for PCR is pAMG2'-murine
OPG binding protein [158-316] below and oligonucleotides #1619-86
and #1581-76 will be the primer pair to be used for PCR and cloning
this gene construct.
TABLE-US-00008 1619-86:
5'-GTTCTCCTCATATGCGTGGTAAACCTGAAGCTCAACCATTTGCA-3' (SEQ ID NO:18)
1581-76: 5'-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID
NO:3)
pAMG21-Murine OPG Binding Protein [158-316]
This construct was engineered to be 160 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met-Lys(158)-Pro-Glu-Ala--------Gln-Asp-Ile-Asp(316)-COOH
(SEQ ID NO: 19). The template to be used for PCR was pcDNA/32D-F3
and oligonucleotides #1581-73 and #1581-76 were the primer pair to
be used for PCR and cloning this gene construct.
TABLE-US-00009 1581-73:
5'-GTTCTCCTCATATGAAACCTGAAGCTCAACCATTTGCACACCTCACCATCAAT-3' (SEQ ID
NO:20) 1581-76: 5'-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID
NO:3)
pAMG21-Murine OPG Binding Protein [166-316]
This construct is engineered to be 152 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met-His(166)-Leu-Thr-Ile--------Gln-Asp-Ile-Asp(316)-COOH
(SEQ ID NO: 21) The template to be used for PCR is pcDNA/32D-F3 and
oligonucleotides #1581-75 and #1581-76 will be the primer pair to
be used for PCR and cloning this gene construct.
TABLE-US-00010 1581-75:
5'-GTTCTCCTCATATGCATTTAACTATTACGCTGCATCTATCCCAT
CGGGTTCCCATAAAGTCACT-3' (SEQ ID NO:22) 1581-76:
5'-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID NO:3)
pAMG21-Murine OPG Binding Protein [168-316]
This construct is engineered to be 150 amino acids in length and
have the following N-terminal and C-terminal residues,
NH.sub.2-Met-Thr(168)-Ile-Asn-Ala--------Gln-Asp-Ile-Asp(316)-COOH
(SEQ ID NO: 3). The template to be used for PCR is pcDNA/32D-F3 and
oligonucleotides #1581-74 and #1581-76 will be the primer pair to
be used for PCR and cloning.
TABLE-US-00011 1581-74:
5'-GTTCTCCTCATATGACTATTAACGCTGCATCTATCCCATCGGGTTCCCATAAAGTCACT-3'-
(SEQ ID NO:24) 1581-76: 5'-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3'
(SEQ ID NO:3)
It is understood that the above constructs are examples and one
skilled in the art may readily obtain other forms of OPG binding
protein using the general methodology presented her.
Recombinant bacterial constructs pAMG21-murine OPG binding protein
[75-316], [95-316], [107-316], [118-316], [128-316], [137-316], and
[158-316] have been cloned, DNA sequence confirmed, and levels of
recombinant gene product expression following induction has been
examined. All constructs produced levels of recombinant gene
product which was readily visible following SDS polyacrylamide gel
electrophoresis and coomassie staining of crude lysates. Growth of
transformed E. coli 393 or 2596, induction of OPG binding protein
expression and isolation of inclusion bodies containing OPG binding
protein is done according to procedures described in U.S. Ser. No.
08/577,788 filed Dec. 22, 1995 now U.S. Pat. No. 6,613,544.
Purification of OPG binding proteins from inclusion bodies requires
solubilization and renaturing of OPG binding protein using
procedures available to one skilled in the art. Recombinant murine
OPG binding protein [158-316] was found to be produced mostly
insolubly, but about 40% was found in the soluble fraction.
Recombinant protein was purified from the soluble fraction as
described below and its bioactivity examined.
EXAMPLE 7
Purification of Recombinant Murine OPG Ligand [158-316]
Frozen bacterial cells harboring expressed murine OPG binding
protein (158-316) were thawed and resuspended in 20 mM tris-HCl pH
7.0, 10 mM EDTA. The cell suspension (20% w/v) was then homogenized
by three passes through a microfluidizer. The lysed cell suspension
was centrifuged in a JA14 rotor at 10,000 rpm for 45 minutes.
SDS-PAGE analysis showed a band of approximately 18 kd molecular
weight present in both inclusion bodies and the supernatant. The
soluble fraction was then applied to a Pharmacia SP Sepharose 4FF
column equilibrated with 10 mM MES pH 6.0. The OPG binding protein
was eluted with a 20 column volume gradient of 0 0.4M NaCl in MES
pH 6.0. Fractions containing OPG binding protein were then applied
to an ABX Bakerbond column equilibrated with 20 mM MES pH 6.0. OPG
binding protein was eluted with a 15CV gradient of 0 0.5M NaCl in
MES pH 6.0. The final product was over 95% homogeneous by SDS-PAGE.
N-terminal sequencing gave the following sequence:
Met-Lys-Pro-Glu-Ala-Gln-Pro-Phe-Ala-His (SEQ ID NO: 25) which was
identified to that predicted for a polypeptide starting at residue
158 (with an initiator methionine). The relative molecular weight
of the protein during SDS-PAGE does not change upon reduction.
EXAMPLE 8
In Vitro Bioactivity of Recombinant Soluble OPG-bp
Recombinant OPG protein has previously been shown to block vitamin
D3-dependent osteoclast formation from bone marrow and spleen
precursors in an osteoclast forming assay as described in U.S. Ser.
No. 08/577,788 now U.S. Pat. No. 6,613,544. Since OPG binding
protein binds to OPG, and is a novel member of the TNF family of
ligands, it is a potential target of OPG bioactivity. Recombinant
soluble OPG binding protein (158-316), representing the minimal
core TNF.alpha.-like domain, was tested for its ability to modulate
osteoclast differentiation from osteoclast precursors. Bone marrow
cells were isolated from adult mouse femurs, and treated with
M-CSF. The non-adherent fraction was co-cultured with ST2 cells in
the presence and absence of both vitamin D3 and dexamethasone. As
previously shown, osteoclasts develop only from co-cultures
containing stromal cells (ST2), vitamin D3 and dexamethasone.
Recombinant soluble OPG binding protein was added at varying
concentrations ranging from 0.16 to 500 ng/ml and osteoclast
maturation was determined by TRAP solution assay and by visual
observation. OPG binding protein strongly stimulated osteoclast
differentiation and maturation in a dose dependent manner, with
half-maximal effects in the 1 2 ng/ml range, suggesting that it
acts as an potent inducer of osteoclastogenesis in vitro (FIG. 5).
The effect of OPG binding protein is blocked by recombinant OPG
(FIG. 6).
To test whether OPG binding protein could replace the stroma and
added steroids, cultures were established using M-CSF at varying
concentrations to promote the growth of osteoclast precursors and
various amounts of OPG binding protein were also added. As shown in
FIG. 6, OPG binding protein dose dependently stimultated TRAP
activity, and the magnitude of the stimulation was dependent on the
level of added M-CSF suggesting that these two factors together are
pivotal for osteoclast development. To confirm the biological
relevance of this last observation, cultures were established on
bovine cortical bone slices and the effects of M-CSF and OPG
binding protein either alone or together were tested. As shown in
FIG. 7, OPG binding protein in the presence of M-CSF stimulated the
formation of large TRAP positive osteoclasts that eroded the bone
surface resulting in pits. Thus, OPG binding protein acts as an
osteoclastogenesis stimulating (differentiation) factor. This
suggests that OPG blocks osteoclast development by sequestering OPG
binding protein.
EXAMPLE 9
In Vivo Activity of Recombinant Soluble OPG Binding Protein
Based on in vitro studies, recombinant murine OPG binding protein
[158-316] produced in E. coli is a potent inducer of osteoclast
development from myeloid precursors. To determine its effects in
vivo, male BDF1 mice aged 4 5 weeks (Charles River Laboratories)
received subcutaneous injections of OPG binding protein [158-316]
twice a day for three days and on the morning of the fourth day
(days 0, 1, 2, and 3). Five groups of mice (n=4) received carrier
alone, or 1, 5, 25 or 100 .mu.g/of of OPG binding protein [158-316]
per day. An additional 5 groups of mice (n=4) received the above
doses of carrier or of OPG binding protein [158-316] and in
addition received human Fc-OPG [22-194] at 1 mg/Kg/day
(approximately 20 .mu.g/day) by single daily subcutaneous
injection. Whole blood ionized calcium was determined prior to
treatment on day 0 and 3 4 hours after the first daily injection of
of OPG binding protein [158-316] on days 1, 2, and 3. Four hours
after the last injection on day 3 the mice were sacrificed and
radiographs were taken.
Recombinant of OPG binding protein [158-316] produced a significant
increase in blood ionized calcium after two days of treament at
dose of 5 .mu.g/day and higher (FIG. 8). The severity of the
hypercalcemia indicates a potent induction of osteoclast activity
resulting from increased bone resorption. Concurrent OPG
administration limited hypercalcemia at doses of OPG binding
protein [158-316] of 5 and 25 .mu.g/day, but not at 100 .mu.g/day.
These same animal were analysed by radiaography to determine if
there were any effects on bone mineral density visible by X-ray
(FIG. 9). Recombinant of OPG binding protein [158-316] injected for
3 days decreased bone density in the proximal tibia of mice in a
dose-dependent manner. The reduction in bone density was
particularly evident in mice receiving 100 .mu.g/d confirming that
the profound hypercalcemia in these animals was produced from
increased bone resorption and the resulting release of calcium from
the skeleton. These data clearly indicate that of OPG binding
protein [158-316] acts in vivo to promote bone resorption, leading
to systemic hypercalcemia, and recombinant OPG abbrogates these
effects.
EXAMPLE 10
Cloning and Expression of Soluble OPG Binding Protein in Mammalian
Cells
The full length clone of murine and human OPG binding protein can
be expressed in mammalian cells as previously described in Example
2. Alternatively, the cDNA clones can be modified to encode
secreted forms of the protein when expressed in mammalian cells. To
do this, the natural 5'-end of the cDNA encoding the intiation
codon, and extending approximately through the first 69 amino acid
of the protein, including the transmembrane spanning region, could
be replaced with a signal peptide leader sequence. For example, DNA
sequences encoding the initiation codon and signal peptide of a
known gene can be spliced to the OPG binding protein cDNA sequence
beginning anywhere after the region encoding amino acid residue 68.
The resulting recombinant clones are predicted to produce secreted
forms of OPB binding protein in mammalian cells, and should undergo
post translational modifications which normally occur in the
C-terminal extracellular domain of OPG binding protein, such as
glycoslyation. Using this strategy, a secreted form of OPG binding
protein was constructed which has at its 5' end the murine OPG
signal peptide, and at its 3' end the human IgG1 Fc domain. The
plasmid vector pCEP4/muOPG[22-401]-Fc as described in U.S. Ser. No.
08/577,788, filed Dec. 22, 1995, now U.S. Pat. No. 6,613,544, was
digested with NotI to cleave between the 3' end of OPG and the Fc
gene. The linearized DNA was then partially digested with XmnI to
cleave only between residues 23 and 24 of OPG leaving a blunt end.
The restriction digests were then dephosphorylated with CIP and the
vector portion of this digest (including residues 1 23 of OPG and
Fc) was gel purified.
The murine OPG binding protein cDNA region encoding amino acid
reisudes 69 316 were PCR amplified using Pfu Polymerase
(Stratagene, San Diego, Calif.) from the plasmid template using
primers the following oligonucleotides:
TABLE-US-00012 1602-61: CCT CTA GGC CTG TAC TTT CGA GCG CAG ATG
(SEQ ID NO:26) 1602-59: CCT CTG CGG CCG CGT CTA TGT CCT GAA CTT TG
(SEQ ID NO:27)
The 1602-61 oligonucleotide amplifies the 5' end of the gene and
contains an artificial an StuI site. The 1602-59 primer amplifies
the 3' end of the gene and contains an artificial NotI site. The
resulting PCR product obtained was digested with NotI and StuI,
then gel purified. The purified PCR product was ligated with
vector, then used to transform electrocompetent E. coli DH10B
cells. The resulting clone was sequenced to confirm the integrity
of the amplified sequence and restriction site junctions. This
plasmid was then used to transfect human 293 fibroblasts, and the
OPG binding protein-Fc fusion protein was collected form culture
media as previously described in U.S. Ser. No. 08/577,788, filed
Dec. 22, 1995 now U.S. Pat. No. 6,613,544.
Using a similar strategy, an expression vector was designed that is
capable of expressing a N-terminal truncation of fused to the human
IgG1 Fc domain. This construct consists of the murine OPG signal
peptide (aa residue 1 21), fused in frame to murine OPG binding
protein residues 158 316, followed by an inframe fusion to human
IgG1 Fc domain. To do this, the plasmid vector pCEP4/murine OPG
[22-401] (U.S. Ser. No. 08/577,788, filed Dec. 22, 1995, now U.S.
Pat. No. 6,613,544), was digested with HindIII and NotI to remove
the entire OPG reading frame. Murine OPG binding protein, residues
158 316 were PCR amplified using from the plasmid template
pCDNA/32D-F3 using the following primers:
TABLE-US-00013 1616-44: CCT CTC TCG AGT GGA CAA CCC AGA AGC CTG AGG
CCC AGC CAT TTG C 1602-59: CCT CTG CGG CCG CGT CTA TGT CCT GAA CTT
TG
1616-44 amplifies OPG binding protein starting at residue 158 as
well as containing residues 16 21 of the muOPG signal peptide with
an artificial XhoI site. 1602-59 amplifies the 3' end of the gene
and adds an in-frame NotI site. The PCR product was digested with
NotI and XhoI and then gel purified.
The Follwing complimentary primers were annealed to each other to
form an adapter encoding the murine OPG signal peptide and Kozak
sequence surrounding the translation initiation site:
TABLE-US-00014 1616-41: AGC TTC CAC CAT GAA CAA GTG GCT GTG CTG CGC
ACT CCT GGT GCT CCT GGA CAT CA (SEQ ID NO:30) 1616-42: TCG ATG ATG
TCC AGG AGC ACC AGG AGT GCG CAG CAC AGC CAC TTG TTC ATG GTG GA (SEQ
ID NO:31)
These primers were annealed, generating 5' overhangs compatible
with HindIII on the 5' end and XhoI on the 3' end. The digested
vector obtianed above, the annealed oligos, and the digested PCR
fragment were ligated together and electroporated into DH10B cells.
The resulting clone was sequenced to confirm authentic
reconstruction of the junction between the signal peptide, OPG
binding protein fragment encoding residues 158 316, and the IgG1 Fc
domain. The recombiant plasmid was purified, transfected into human
293 fibroblasts, and expressed as a conditioned media product as
described above.
EXAMPLE 11
Peptides of the OPG Binding Protein and Preparation of Polyclonal
and Monoclonal Antibodies to the Protein
Antibodies to specific regions of the OPG binding protein may be
obtained by immunization with peptides from OPG binding protein.
These peptides may be used alone, or conjugated forms of the
peptide may be used for immunization.
The crystal structure of mature TNF.alpha. has been described [E.
Y. Jones, D. I. Stuart, and N. P. C. Walker (1990) J. Cell Sci.
Suppl. 13, 11 18] and the monomer forms an antiparallel
.beta.-pleated sheet sandwich with a jellyroll topology. Ten
antiparallel .beta.-strands are observed in this crystal structure
and form a beta sandwich with one beta sheet consisting of strands
B'BIDG and the other of strands C'CHEF [E. Y. Jones et al., ibid.]
Two loops of mature TNF.alpha. have been implicated from
mutagenesis studies to make contacts with receptor, these being the
loops formed between beta strand B & B' and the loop between
beta strands E & F [C. R. Goh, C-S. Loh, and A. G. Porter
(1991) Protein Engineering 4, 785 791]. The crystal structure of
the complex formed between TNF.beta. and the extracellular domain
of the 55 kd TNF receptor (TNF-R55) has been solved and the
receptor-ligand contacts have been described [D. W. Banner, A.
D'Arcy, W. Janes, R. Gentz, H-J. Schoenfeld, C. Broger, H.
Loetscher, and W. Lesslauer (1993) Cell 73, 431 445]. In agreement
with mutagenesis studies described above [C. R. Goh et al., ibid.]
the corresponding loops BB' and EF of the ligand TNF.beta. were
found to make the majority of contacts with the receptor in the
resolved crystal structure of the TNFb:TNF-R55 complex. The amino
acid sequence of murine OPG binding protein was compared to the
amino acid sequences of TNF.alpha. and TNF.beta.. The regions of
murine OPG binding protein corresponding to the BB' and EF loops
were predicted based on this comparison and peptides have been
designed and are described below
A. Antigen(s): Recombinant murine OPG binding protein [158-316] has
been used as an antigen (ag) for immunization of animals as
described below, and serum will be examined using approaches
described below. Peptides to the putative BB' and EF loops of
murine OPG binding protein have been synthesized and will be used
for immunization; these peptides are:
TABLE-US-00015 BB' loop peptide:
NH2--NAASIPSGSHKVTLSSWYHDRGWAKIS--COOH (SEQ ID NO:32) BB' loop-Cys
peptide: NH2--NAASIPSGSHKVTLSSWYHDRGWAKISC--COOH (SEQ ID NO:33) EF
loop peptide: NH2--VYVVKTSIKIPSSHNLM--COOH (SEQ ID NO:34) EF
loop-Cys peptide: NH2--VYVVKTSIKIPSSHNLMC--COOH (SEQ ID NO:35)
Peptides with a carboxy-terminal cysteine residue have been used
for conjugation using approaches described in section B below, and
have been used for immunization.
B. Keyhole Limpet Hemocyanin or Bovine Serum Albumin Conjugation:
Selected peptides or protein fragments may be conjugated to keyhole
limpet hemocyanin (KLH) in order to increase their immunogenicity
in animals. Also, bovine serum albumin (BSA) conjugated peptides or
protein fragments may be utilized in the EIA protocol. Imject
Maleimide Activated KLH or BSA (Pierce Chemical Company, Rockford,
Ill.) is reconstituted in dH.sub.2 O to a final concentration of 10
mg/ml. Peptide or protein fragments are dissolved in phosphate
buffer then mixed with an equivalent mass (g/g) of KLH or BSA. The
conjugation is allowed to react for 2 hours at room temperature
(rt) with gentle stirring. The solution is then passed over a
desalting column or dialyzed against PBS overnight. The peptide
conjugate is stored at -20.degree. C. until used in immunizations
or in EIAs.
C. Immunization: Balb/c mice, (Charles Rivers Laboratories,
Wilmington, Mass.) Lou rats, or New Zealand White rabbits will be
subcutaneously injected (SQI) with ag (50 .mu.g, 150 .mu.g, and 100
.mu.g respectively) emulsified in Complete Freund's Adjuvant (CFA,
50% vol/vol; Difco Laboratories, Detroit, Mich.). Rabbits are then
boosted two or three times at 2 week intervals with antigen
prepared in similar fashion in Incomplete Freund's Adjuvant (ICFA;
Difco Laboratories, Detroit, Mich.). Mice and rats are boosted
approximately every 4 weeks. Seven days following the second boost,
test bleeds are performed and serum antibody titers determined.
When a titer has developed in rabbits, weekly production bleeds of
50 mls are taken for 6 consecutive weeks. Mice and rats are
selected for hybridoma production based on serum titer levels;
animals with half-maximal titers greater than 5000 are used.
Adjustments to this protocol may be applied by one skilled in the
art; for example, various types of immunomodulators are now
available and may be incorporated into this protocol.
D. Enzyme-linked Immunosorbent Assay (EIA): EIAs will be performed
to determine serum antibody (ab) titres of individual animals, and
later for the screening of potential hybridomas. Flat bottom,
high-binding, 96-well microtitration EIA/RIA plates (Costar
Corporation, Cambridge, Mass.) will be coated with purified
recombinant protein or protein fragment (antigen, ag) at 5 .mu.g
per ml in carbonate-bicarbonate buffer, pH 9.2 (0.015 M
Na.sub.2CO.sub.3, 0.035 M NaHCO.sub.3). Protein fragments may be
conjugated to bovine serum albumin (BSA) if necessary. Fifty 1
.mu.l of ag will be added to each well. Plates will then be covered
with acetate film (ICN Biomedicals, Inc., Costa Mesa, Calif.) and
incubated at room temperature (rt) on a rocking platform for 2
hours or over-night at 4.degree. C. Plates will be blocked for 30
minutes at rt with 250 .mu.l per well 5% BSA solution prepared by
mixing 1 part BSA diluent/blocking solution concentrate (Kirkegaard
and Perry Laboratories, Inc., Gaithersburg, Md.) with 1 part
deionized water (dH.sub.2O). Blocking solution having been
discarded, 50 .mu.l of serum 2-fold dilutions (1:100 through
1:12,800) or hybridoma tissue culture supernatants will be added to
each well. Serum diluent is 1% BSA (10% BSA diluent/blocking
solution concentrate diluted 1:10 in Dulbecco's Phosphate Buffered
Saline, D-PBS; Gibco BRL, Grand Island, N.Y.)) while hybridoma
supernatants are tested undiluted. In the case of hybridoma
screening, one well is maintained as a conjugate control, and a
second well as a positive ab control. Plates are again incubated at
rt, rocking for 1 hour, then washed 4 times using a 1.times.
preparation of wash solution 20.times. concentrate (Kirkegaard and
Perry Laboratories, Inc., Gaithersburg, Md.) in dH.sub.2O.
Horseradish peroxidase conjugated secondary ab (Boeringer Mannheim
Biochemicals, Indianapolis, Ind.) diluted in 1% BSA is then
incubated in each well for 30 minutes. Plates are washed as before,
blotted dry, and ABTS peroxidase single component substrate
(Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.) is
added. Absorbance is read at 405 nm for each well using a
Microplate EL310 reader (Bio-tek Instruments, Inc., Winooski, Vt.).
Half-maximal titre of serum antibody is calculated by plotting the
log.sub.10 of the serum dilution versus the optical density at 405,
then extrapolating at the 50% point of the maximal optical density
obtained by that serum. Hybridomas are selected as positive if
optical density scores greater than 5-fold above background.
Adjustments to this protocol may be applied; in example, conjugated
secondary antibody may be chosen for specificity or
non-cross-reactivity.
E. Cell fusion: The animal selected for hybridoma production is
intravenously injected with 50 to 100 .mu.g of ag in PBS. Four days
later, the animal is sacrificed by carbon dioxide and its spleen
collected under sterile conditions into 35 ml Dulbeccos' Modified
Eagle's Medium containing 200 U/ml Penicillin G, 200 .mu.g/ml
Streptomycin Sulfate, and 4 mM glutamine (2.times. P/S/G DMEM). The
spleen is trimmed of excess fatty tissue, then rinsed through 4
dishes of clean 2.times. P/S/G DMEM. It is next transferred to a
sterile stomacher bag (Tekmar, Cincinnati, Ohio) containing 10 ml
of 2.times. P/S/G DMEM and disrupted to single cell suspension with
the Stomacher Lab Blender 80 (Seward Laboratory UAC House; London,
England). As cells are released from the spleen capsule into the
media, they are removed from the bag and transferred to a sterile
50 ml conical centrifuge tube (Becton Dickinson and Company,
Lincoln Park, N.J.). Fresh media is added to the bag and the
process is continued until the entire cell content of the spleen is
released. These splenocytes are washed 3 times by centrifugation at
225.times.g for 10 minutes.
Concurrently, log phase cultures of myeloma cells, Sp2/0-Ag14 or
Y3-Ag1.2.3 for mouse or rat splenocyte fusions, respectively,
(American Type Culture Collection; 10801 University Boulevard,
Manassas, Va. 20110-2209) grown in complete medium (DMEM, 10%
inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM
non-essential amino acids, 1 mM sodium pyruvate, and 10 mM hepes
buffer; Gibco Laboratories, Grand Island, N.Y.) are washed in
similar fashion. The splenocytes are combined with the myeloma
cells and pelleted once again. The media is aspirated from the cell
pellet and 2 ml of polyethylene glycol 1500 (PEG 1500; Boehringer
Mannheim Biochemicals, Indianapolis, Ind.) is gently mixed into the
cells over the course of 1 minute. Thereafter, an equal volume of
2.times. P/S/G DMEM is slowly added. The cells are allowed to fuse
at 370.degree. C. for 2 minutes, then an additional 6 ml of
2.times. P/S/G DMEM is added. The cells are again set at 37.degree.
C. for 3 minutes. Finally, 35 ml of 2.times. P/S/G DMEM is added to
the cell suspension, and the cells pelleted by centrifugation.
Media is aspirated from the pellet and the cells gently resuspended
in complete medium. The cells are distributed over 96-well
flat-bottom tissue culture plates (Becton Dickinson Labware;
Lincoln Park, N.J.) by single drops from a 5 ml pipette. Plates are
incubated overnight in humidified conditions at 37.degree. C., 5%
CO.sub.2. The next day, an equal volume of selection medium is
added to each well. Selection consists of 0.1 mM hypoxanthine,
4.times.10.sup.-4 mM aminopterin, and 1.6.times.10.sup.-2 mM
thymidine in complete medium. The fusion plates are incubated for 7
days followed by 2 changes of medium during the next 3 days; HAT
selection medium is used after each fluid change. Tissue culture
supernatants are taken 3 to 4 days after the last fluid change from
each hybrid-containing well and tested by EIA for specific antibody
reactivity. This protocol has been modified by that in Hudson and
Hay, "Practical Immunology, Second Edition", Blackwell Scientific
Publications.
While the present invention has been described in terms of the
preferred embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Therefore, it
is intended that the appended claims cover all such equivalent
variations which come within the scope of the invention as
claimed.
SEQUENCE LISTINGS
1
398 amino acidsamino acidsinglelinearprotein 1Asp Pro Asn Arg Gln
Asp Ile Asp1 552 base pairsnucleic acidsinglelinearcDNA 2GTTCTCCTCA
TATGGATCCA AACCGTATTT CTGAAGACAG CACTCACTGC TT 5237 base
pairsnucleic acidsinglelinearcDNA 3TACGCACTCC GCGGTTAGTC TATGTCCTGA
ACTTTGA 378 amino acidsamino acidsinglelinearprotein 4Glu Asn Ala
Gly Gln Asp Ile Asp1 551 base pairsnucleic acidsinglelinearcDNA
5ATTTGATTCT AGAAGGAGGA ATAACATATG CATGAAAACG CAGGTCTGCA G 5142 base
pairsnucleic acidsinglelinearcDNA 6TATCCGCGGA TCCTCGAGTT AGTCTATGTC
CTGAACTTTG AA 428 amino acidsamino acidsinglelinearprotein 7Glu Asp
Thr Leu Gln Asp Ile Asp1 554 base pairsnucleic acidsinglelinearcDNA
8ATTTGATTCT AGAAGGAGGA ATAACATATG TCTGAAGACA CTCTGCCGGA CTCC 549
amino acidsamino acidsinglelinearprotein 9Lys Gln Ala Phe Gln Gln
Asp Ile Asp1 548 base pairsnucleic acidsinglelinearcDNA
10ATTTGATTCT AGAAGGAGGA ATAACATATG AAACAAGCTT TTCAGGGG 4810 amino
acidsamino acidsinglelinearprotein 11Met Lys Glu Leu Gln His Gln
Asp Ile Asp1 5 1051 base pairsnucleic acidsinglelinearcDNA
12ATTTGATTCT AGAAGGAGGA ATAACATATG AAAGAACTGC AGCACATTGT G 5110
amino acidsamino acidsinglelinearprotein 13Met Gln Arg Phe Ser Gly
Gln Asp Ile Asp1 5 1051 base pairsnucleic acidsinglelinearcDNA
14ATTTGATTCT AGAAGGAGGA ATAACATATG CAGCGTTTCT CTGGTGCTCC A 519
amino acidsamino acidsinglelinearprotein 15Met Glu Gly Ser Trp Gln
Asp Ile Asp1 540 base pairsnucleic acidsinglelinearcDNA
16GTTCTCCTCA TATGGAAGGT TCTTGGTTGG ATGTGGCCCA 409 amino acidsamino
acidsinglelinearprotein 17Met Arg Gly Lys Pro Gln Asp Ile Asp1 544
base pairsnucleic acidsinglelinearcDNA 18GTTCTCCTCA TATGCGTGGT
AAACCTGAAG CTCAACCATT TGCA 449 amino acidsamino
acidsinglelinearprotein 19Met Lys Pro Glu Ala Gln Asp Ile Asp1 553
base pairsnucleic acidsinglelinearcDNA 20GTTCTCCTCA TATGAAACCT
GAAGCTCAAC CATTTGCACA CCTCACCATC AAT 539 amino acidsamino
acidsinglelinearprotein 21Met His Leu Thr Ile Gln Asp Ile Asp1 565
base pairsnucleic acidsinglelinearcDNA 22GTTCTCCTCA TATGCATTTA
ACTATTAACG CTGCATCTAT CCCATCGGGT TCCCATAAAG 60TCACT 659 amino
acidsamino acidsinglelinearprotein 23Met Thr Ile Asn Ala Gln Asp
Ile Asp1 559 base pairsnucleic acidsinglelinearcDNA 24GTTCTCCTCA
TATGACTATT AACGCTGCAT CTATCCCATC GGGTTCCCAT AAAGTCACT 5910 amino
acidsamino acidsinglelinearprotein 25Met Lys Pro Glu Ala Gln Pro
Phe Ala His1 5 1030 base pairsnucleic acidsinglelinearcDNA
26CCTCTAGGCC TGTACTTTCG AGCGCAGATG 3032 base pairsnucleic
acidsinglelinearcDNA 27CCTCTGCGGC CGCGTCTATG TCCTGAACTT TG 3246
base pairsnucleic acidsinglelinearcDNA 28CCTCTCTCGA GTGGACAACC
CAGAAGCCTG AGGCCCAGCC ATTTGC 4632 base pairsnucleic
acidsinglelinearcDNA 29CCTCTGCGGC CGCGTCTATG TCCTGAACTT TG 3256
base pairsnucleic acidsinglelinearcDNA 30AGCTTCCACC ATGAACAAGT
GGCTGTGCTG CGCACTCCTG GTGCTCCTGG ACATCA 5656 base pairsnucleic
acidsinglelinearcDNA 31TCGATGATGT CCAGGAGCAC CAGGAGTGCG CAGCACAGCC
ACTTGTTCAT GGTGGA 5627 amino acidsamino acidsinglelinearprotein
32Asn Ala Ala Ser Ile Pro Ser Gly Ser His Lys Val Thr Leu Ser Ser1
5 10 15Trp Tyr His Asp Arg Gly Trp Ala Lys Ile Ser 20 2528 amino
acidsamino acidsinglelinearprotein 33Asn Ala Ala Ser Ile Pro Ser
Gly Ser His Lys Val Thr Leu Ser Ser1 5 10 15Trp Tyr His Asp Arg Gly
Trp Ala Lys Ile Ser Cys 20 2517 amino acidsamino
acidsinglelinearprotein 34Val Tyr Val Val Lys Thr Ser Ile Lys Ile
Pro Ser Ser His Asn Leu1 5 10 15Met18 amino acidsamino
acidsinglelinearprotein 35Val Tyr Val Val Lys Thr Ser Ile Lys Ile
Pro Ser Ser His Asn Leu1 5 10 15Met Cys2295 base pairsnucleic
acidsinglelinearcDNACDS 158..1105 36GAGCTCGGAT CCACTACTCG
ACCCACGCGT CCGGCCAGGA CCTCTGTGAA CCGGTCGGGG 60CGGGGGCCGC CTGGCCGGGA
GTCTGCTCGG CGGTGGGTGG CCGAGGAAGG GAGAGAACGA 120TCGCGGAGCA
GGGCGCCCGA ACTCCGGGCG CCGCGCC ATG CGC CGG GCC AGC CGA 175 Met Arg
Arg Ala Ser Arg 1 5GAC TAC GGC AAG TAC CTG CGC AGC TCG GAG GAG ATG
GGC AGC GGC CCC 223Asp Tyr Gly Lys Tyr Leu Arg Ser Ser Glu Glu Met
Gly Ser Gly Pro 10 15 20GGC GTC CCA CAC GAG GGT CCG CTG CAC CCC GCG
CCT TCT GCA CCG GCT 271Gly Val Pro His Glu Gly Pro Leu His Pro Ala
Pro Ser Ala Pro Ala 25 30 35CCG GCG CCG CCA CCC GCC GCC TCC CGC TCC
ATG TTC CTG GCC CTC CTG 319Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser
Met Phe Leu Ala Leu Leu 40 45 50GGG CTG GGA CTG GGC CAG GTG GTC TGC
AGC ATC GCT CTG TTC CTG TAC 367Gly Leu Gly Leu Gly Gln Val Val Cys
Ser Ile Ala Leu Phe Leu Tyr 55 60 65 70TTT CGA GCG CAG ATG GAT CCT
AAC AGA ATA TCA GAA GAC AGC ACT CAC 415Phe Arg Ala Gln Met Asp Pro
Asn Arg Ile Ser Glu Asp Ser Thr His 75 80 85TGC TTT TAT AGA ATC CTG
AGA CTC CAT GAA AAC GCA GGT TTG CAG GAC 463Cys Phe Tyr Arg Ile Leu
Arg Leu His Glu Asn Ala Gly Leu Gln Asp 90 95 100TCG ACT CTG GAG
AGT GAA GAC ACA CTA CCT GAC TCC TGC AGG AGG ATG 511Ser Thr Leu Glu
Ser Glu Asp Thr Leu Pro Asp Ser Cys Arg Arg Met 105 110 115AAA CAA
GCC TTT CAG GGG GCC GTG CAG AAG GAA CTG CAA CAC ATT GTG 559Lys Gln
Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile Val 120 125
130GGG CCA CAG CGC TTC TCA GGA GCT CCA GCT ATG ATG GAA GGC TCA TGG
607Gly Pro Gln Arg Phe Ser Gly Ala Pro Ala Met Met Glu Gly Ser
Trp135 140 145 150TTG GAT GTG GCC CAG CGA GGC AAG CCT GAG GCC CAG
CCA TTT GCA CAC 655Leu Asp Val Ala Gln Arg Gly Lys Pro Glu Ala Gln
Pro Phe Ala His 155 160 165CTC ACC ATC AAT GCT GCC AGC ATC CCA TCG
GGT TCC CAT AAA GTC ACT 703Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser
Gly Ser His Lys Val Thr 170 175 180CTG TCC TCT TGG TAC CAC GAT CGA
GGC TGG GCC AAG ATC TCT AAC ATG 751Leu Ser Ser Trp Tyr His Asp Arg
Gly Trp Ala Lys Ile Ser Asn Met 185 190 195ACG TTA AGC AAC GGA AAA
CTA AGG GTT AAC CAA GAT GGC TTC TAT TAC 799Thr Leu Ser Asn Gly Lys
Leu Arg Val Asn Gln Asp Gly Phe Tyr Tyr 200 205 210CTG TAC GCC AAC
ATT TGC TTT CGG CAT CAT GAA ACA TCG GGA AGC GTA 847Leu Tyr Ala Asn
Ile Cys Phe Arg His His Glu Thr Ser Gly Ser Val215 220 225 230CCT
ACA GAC TAT CTT CAG CTG ATG GTG TAT GTC GTT AAA ACC AGC ATC 895Pro
Thr Asp Tyr Leu Gln Leu Met Val Tyr Val Val Lys Thr Ser Ile 235 240
245AAA ATC CCA AGT TCT CAT AAC CTG ATG AAA GGA GGG AGC ACG AAA AAC
943Lys Ile Pro Ser Ser His Asn Leu Met Lys Gly Gly Ser Thr Lys Asn
250 255 260TGG TCG GGC AAT TCT GAA TTC CAC TTT TAT TCC ATA AAT GTT
GGG GGA 991Trp Ser Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn Val
Gly Gly 265 270 275TTT TTC AAG CTC CGA GCT GGT GAA GAA ATT AGC ATT
CAG GTG TCC AAC 1039Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile Ser Ile
Gln Val Ser Asn 280 285 290CCT TCC CTG CTG GAT CCG GAT CAA GAT GCG
ACG TAC TTT GGG GCT TTC 1087Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala
Thr Tyr Phe Gly Ala Phe295 300 305 310AAA GTT CAG GAC ATA GAC
TGAGACTCAT TTCGTGGAAC ATTAGCATGG 1135Lys Val Gln Asp Ile Asp
315ATGTCCTAGA TGTTTGGAAA CTTCTTAAAA AATGGATGAT GTCTATACAT
GTGTAAGACT 1195ACTAAGAGAC ATGGCCCACG GTGTATGAAA CTCACAGCCC
TCTCTCTTGA GCCTGTACAG 1255GTTGTGTATA TGTAAAGTCC ATAGGTGATG
TTAGATTCAT GGTGATTACA CAACGGTTTT 1315ACAATTTTGT AATGATTTCC
TAGAATTGAA CCAGATTGGG AGAGGTATTC CGATGCTTAT 1375GAAAAACTTA
CACGTGAGCT ATGGAAGGGG GTCACAGTCT CTGGGTCTAA CCCCTGGACA
1435TGTGCCACTG AGAACCTTGA AATTAAGAGG ATGCCATGTC ATTGCAAAGA
AATGATAGTG 1495TGAAGGGTTA AGTTCTTTTG AATTGTTACA TTGCGCTGGG
ACCTGCAAAT AAGTTCTTTT 1555TTTCTAATGA GGAGAGAAAA ATATATGTAT
TTTTATATAA TGTCTAAAGT TATATTTCAG 1615GTGTAATGTT TTCTGTGCAA
AGTTTTGTAA ATTATATTTG TGCTATAGTA TTTGATTCAA 1675AATATTTAAA
AATGTCTCAC TGTTGACATA TTTAATGTTT TAAATGTACA GATGTATTTA
1735ACTGGTGCAC TTTGTAATTC CCCTGAAGGT ACTCGTAGCT AAGGGGGCAG
AATACTGTTT 1795CTGGTGACCA CATGTAGTTT ATTTCTTTAT TCTTTTTAAC
TTAATAGAGT CTTCAGACTT 1855GTCAAAACTA TGCAAGCAAA ATAAATAAAT
AAAAATAAAA TGAATACCTT GAATAATAAG 1915TAGGATGTTG GTCACCAGGT
GCCTTTCAAA TTTAGAAGCT AATTGACTTT AGGAGCTGAC 1975ATAGCCAAAA
AGGATACATA ATAGGCTACT GAAATCTGTC AGGAGTATTT ATGCAATTAT
2035TGAACAGGTG TCTTTTTTTA CAAGAGCTAC AAATTGTAAA TTTTGTTTCT
TTTTTTTCCC 2095ATAGAAAATG TACTATAGTT TATCAGCCAA AAAACAATCC
ACTTTTTAAT TTAGTGAAAG 2155TTATTTTATT ATACTGTACA ATAAAAGCAT
TGTCTCTGAA TGTTAATTTT TTGGTACAAA 2215AAATAAATTT GTACGAAAAC
CTGAAAAAAA AAAAAAAAAA AAAAAAAAGG GCGGCCGCTC 2275TAGAGGGCCC
TATTCTATAG 2295316 amino acidsamino acidlinearprotein 37Met Arg Arg
Ala Ser Arg Asp Tyr Gly Lys Tyr Leu Arg Ser Ser Glu 1 5 10 15Glu
Met Gly Ser Gly Pro Gly Val Pro His Glu Gly Pro Leu His Pro 20 25
30Ala Pro Ser Ala Pro Ala Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser
35 40 45Met Phe Leu Ala Leu Leu Gly Leu Gly Leu Gly Gln Val Val Cys
Ser 50 55 60Ile Ala Leu Phe Leu Tyr Phe Arg Ala Gln Met Asp Pro Asn
Arg Ile 65 70 75 80Ser Glu Asp Ser Thr His Cys Phe Tyr Arg Ile Leu
Arg Leu His Glu 85 90 95Asn Ala Gly Leu Gln Asp Ser Thr Leu Glu Ser
Glu Asp Thr Leu Pro 100 105 110Asp Ser Cys Arg Arg Met Lys Gln Ala
Phe Gln Gly Ala Val Gln Lys 115 120 125Glu Leu Gln His Ile Val Gly
Pro Gln Arg Phe Ser Gly Ala Pro Ala 130 135 140Met Met Glu Gly Ser
Trp Leu Asp Val Ala Gln Arg Gly Lys Pro Glu145 150 155 160Ala Gln
Pro Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser 165 170
175Gly Ser His Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly Trp
180 185 190Ala Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg
Val Asn 195 200 205Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys
Phe Arg His His 210 215 220Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr
Leu Gln Leu Met Val Tyr225 230 235 240Val Val Lys Thr Ser Ile Lys
Ile Pro Ser Ser His Asn Leu Met Lys 245 250 255Gly Gly Ser Thr Lys
Asn Trp Ser Gly Asn Ser Glu Phe His Phe Tyr 260 265 270Ser Ile Asn
Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile 275 280 285Ser
Ile Gln Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala 290 295
300Thr Tyr Phe Gly Ala Phe Lys Val Gln Asp Ile Asp305 310 3152271
base pairsnucleic acidsinglelinearcDNACDS 185..1135 38AAGCTTGGTA
CCGAGCTCGG ATCCACTACT CGACCCACGC GTCCGCGCGC CCCAGGAGCC 60AAAGCCGGGC
TCCAAGTCGG CGCCCCACGT CGAGGCTCCG CCGCAGCCTC CGGAGTTGGC
120CGCAGACAAG AAGGGGAGGG AGCGGGAGAG GGAGGAGAGC TCCGAAGCGA
GAGGGCCGAG 180CGCC ATG CGC CGC GCC AGC AGA GAC TAC ACC AAG TAC CTG
CGT GGC TCG 229 Met Arg Arg Ala Ser Arg Asp Tyr Thr Lys Tyr Leu Arg
Gly Ser 1 5 10 15GAG GAG ATG GGC GGC GGC CCC GGA GCC CCG CAC GAG
GGC CCC CTG CAC 277Glu Glu Met Gly Gly Gly Pro Gly Ala Pro His Glu
Gly Pro Leu His 20 25 30GCC CCG CCG CCG CCT GCG CCG CAC CAG CCC CCC
GCC GCC TCC CGC TCC 325Ala Pro Pro Pro Pro Ala Pro His Gln Pro Pro
Ala Ala Ser Arg Ser 35 40 45ATG TTC GTG GCC CTC CTG GGG CTG GGG CTG
GGC CAG GTT GTC TGC AGC 373Met Phe Val Ala Leu Leu Gly Leu Gly Leu
Gly Gln Val Val Cys Ser 50 55 60GTC GCC CTG TTC TTC TAT TTC AGA GCG
CAG ATG GAT CCT AAT AGA ATA 421Val Ala Leu Phe Phe Tyr Phe Arg Ala
Gln Met Asp Pro Asn Arg Ile 65 70 75TCA GAA GAT GGC ACT CAC TGC ATT
TAT AGA ATT TTG AGA CTC CAT GAA 469Ser Glu Asp Gly Thr His Cys Ile
Tyr Arg Ile Leu Arg Leu His Glu 80 85 90 95AAT GCA GAT TTT CAA GAC
ACA ACT CTG GAG AGT CAA GAT ACA AAA TTA 517Asn Ala Asp Phe Gln Asp
Thr Thr Leu Glu Ser Gln Asp Thr Lys Leu 100 105 110ATA CCT GAT TCA
TGT AGG AGA ATT AAA CAG GCC TTT CAA GGA GCT GTG 565Ile Pro Asp Ser
Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly Ala Val 115 120 125CAA AAG
GAA TTA CAA CAT ATC GTT GGA TCA CAG CAC ATC AGA GCA GAG 613Gln Lys
Glu Leu Gln His Ile Val Gly Ser Gln His Ile Arg Ala Glu 130 135
140AAA GCG ATG GTG GAT GGC TCA TGG TTA GAT CTG GCC AAG AGG AGC AAG
661Lys Ala Met Val Asp Gly Ser Trp Leu Asp Leu Ala Lys Arg Ser Lys
145 150 155CTT GAA GCT CAG CCT TTT GCT CAT CTC ACT ATT AAT GCC ACC
GAC ATC 709Leu Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Thr
Asp Ile160 165 170 175CCA TCT GGT TCC CAT AAA GTG AGT CTG TCC TCT
TGG TAC CAT GAT CGG 757Pro Ser Gly Ser His Lys Val Ser Leu Ser Ser
Trp Tyr His Asp Arg 180 185 190GGT TGG GCC AAG ATC TCC AAC ATG ACT
TTT AGC AAT GGA AAA CTA ATA 805Gly Trp Ala Lys Ile Ser Asn Met Thr
Phe Ser Asn Gly Lys Leu Ile 195 200 205GTT AAT CAG GAT GGC TTT TAT
TAC CTG TAT GCC AAC ATT TGC TTT CGA 853Val Asn Gln Asp Gly Phe Tyr
Tyr Leu Tyr Ala Asn Ile Cys Phe Arg 210 215 220CAT CAT GAA ACT TCA
GGA GAC CTA GCT ACA GAG TAT CTT CAA CTA ATG 901His His Glu Thr Ser
Gly Asp Leu Ala Thr Glu Tyr Leu Gln Leu Met 225 230 235GTG TAC GTC
ACT AAA ACC AGC ATC AAA ATC CCA AGT TCT CAT ACC CTG 949Val Tyr Val
Thr Lys Thr Ser Ile Lys Ile Pro Ser Ser His Thr Leu240 245 250
255ATG AAA GGA GGA AGC ACC AAG TAT TGG TCA GGG AAT TCT GAA TTC CAT
997Met Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly Asn Ser Glu Phe His
260 265 270TTT TAT TCC ATA AAC GTT GGT GGA TTT TTT AAG TTA CGG TCT
GGA GAG 1045Phe Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ser
Gly Glu 275 280 285GAA ATC AGC ATC GAG GTC TCC AAC CCC TCC TTA CTG
GAT CCG GAT CAG 1093Glu Ile Ser Ile Glu Val Ser Asn Pro Ser Leu Leu
Asp Pro Asp Gln 290 295 300GAT GCA ACA TAC TTT GGG GCT TTT AAA GTT
CGA GAT ATA GAT 1135Asp Ala Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp
Ile Asp 305 310 315TGAGCCCCAG TTTTTGGAGT GTTATGTATT TCCTGGATGT
TTGGAAACAT TTTTTAAAAC 1195AAGCCAAGAA AGATGTATAT AGGTGTGTGA
GACTACTAAG AGGCATGGCC CCAACGGTAC 1255ACGACTCAGT ATCCATGCTC
TTGACCTTGT AGAGAACACG CGTATTTACA GCCAGTGGGA 1315GATGTTAGAC
TCATGGTGTG TTACACAATG GTTTTTAAAT TTTGTAATGA ATTCCTAGAA
1375TTAAACCAGA TTGGAGCAAT TACGGGTTGA CCTTATGAGA AACTGCATGT
GGGCTATGGG 1435AGGGGTTGGT CCCTGGTCAT GTGCCCCTTC GCAGCTGAAG
TGGAGAGGGT GTCATCTAGC 1495GCAATTGAAG GATCATCTGA AGGGGCAAAT
TCTTTTGAAT TGTTACATCA TGCTGGAACC 1555TGCAAAAAAT ACTTTTTCTA
ATGAGGAGAG AAAATATATG TATTTTTATA TAATATCTAA 1615AGTTATATTT
CAGATGTAAT GTTTTCTTTG CAAAGTATTG TAAATTATAT TTGTGCTATA
1675GTATTTGATT CAAAATATTT AAAAATGTCT TGCTGTTGAC ATATTTAATG
TTTTAAATGT 1735ACAGACATAT TTAACTGGTG CACTTTGTAA ATTCCCTGGG
GAAAACTTGC AGCTAAGGAG 1795GGGAAAAAAA TGTTGTTTCC TAATATCAAA
TGCAGTATAT TTCTTCGTTC TTTTTAAGTT 1855AATAGATTTT TTCAGACTTG
TCAAGCCTGT GCAAAAAAAT TAAAATGGAT GCCTTGAATA 1915ATAAGCAGGA
TGTTGGCCAC CAGGTGCCTT TCAAATTTAG AAACTAATTG ACTTTAGAAA
1975GCTGACATTG CCAAAAAGGA TACATAATGG GCCACTGAAA TCTGTCAAGA
GTAGTTATAT 2035AATTGTTGAA CAGGTGTTTT TCCACAAGTG CCGCAAATTG
TACCTTTTTT TTTTTTTCAA 2095AATAGAAAAG TTATTAGTGG TTTATCAGCA
AAAAAGTCCA ATTTTAATTT AGTAAATGTT 2155ATCTTATACT GTACAATAAA
AACATTGCCT TTGAATGTTA ATTTTTTGGT ACAAAAATAA 2215ATTTATATGA
AAAAAAAAAA AAAAGGGCGG CCGCTCTAGA GGGCCCTATT CTATAG 2271317 amino
acidsamino acidlinearprotein 39Met Arg Arg Ala Ser Arg Asp Tyr Thr
Lys Tyr Leu Arg Gly Ser Glu 1 5 10 15Glu Met Gly Gly Gly Pro Gly
Ala Pro His Glu Gly Pro Leu His Ala 20 25 30Pro Pro Pro Pro Ala Pro
His Gln Pro Pro Ala Ala Ser Arg Ser Met 35 40
45Phe Val Ala Leu Leu Gly Leu Gly Leu Gly Gln Val Val Cys Ser Val
50 55 60Ala Leu Phe Phe Tyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile
Ser 65 70 75 80Glu Asp Gly Thr His Cys Ile Tyr Arg Ile Leu Arg Leu
His Glu Asn 85 90 95Ala Asp Phe Gln Asp Thr Thr Leu Glu Ser Gln Asp
Thr Lys Leu Ile 100 105 110Pro Asp Ser Cys Arg Arg Ile Lys Gln Ala
Phe Gln Gly Ala Val Gln 115 120 125Lys Glu Leu Gln His Ile Val Gly
Ser Gln His Ile Arg Ala Glu Lys 130 135 140Ala Met Val Asp Gly Ser
Trp Leu Asp Leu Ala Lys Arg Ser Lys Leu145 150 155 160Glu Ala Gln
Pro Phe Ala His Leu Thr Ile Asn Ala Thr Asp Ile Pro 165 170 175Ser
Gly Ser His Lys Val Ser Leu Ser Ser Trp Tyr His Asp Arg Gly 180 185
190Trp Ala Lys Ile Ser Asn Met Thr Phe Ser Asn Gly Lys Leu Ile Val
195 200 205Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe
Arg His 210 215 220His Glu Thr Ser Gly Asp Leu Ala Thr Glu Tyr Leu
Gln Leu Met Val225 230 235 240Tyr Val Thr Lys Thr Ser Ile Lys Ile
Pro Ser Ser His Thr Leu Met 245 250 255Lys Gly Gly Ser Thr Lys Tyr
Trp Ser Gly Asn Ser Glu Phe His Phe 260 265 270Tyr Ser Ile Asn Val
Gly Gly Phe Phe Lys Leu Arg Ser Gly Glu Glu 275 280 285Ile Ser Ile
Glu Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp 290 295 300Ala
Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp Ile Asp305 310 315
* * * * *